Genetically-modified immune cells comprising a microRNA-adapted shRNA (shRNAmiR)

ABSTRACT

The present invention encompasses genetically-modified immune cells (and populations thereof) expressing a microRNA-adapted shRNA (shRNAmiR) that reduces the expression of a target endogenous protein. Methods for reducing the expression of an endogenous protein in an immune cell are also provided wherein the method comprises introducing a shRNAmiR that targets the endogenous protein. Using shRNAmiRs for knocking down the expression of a target protein allows for stable knockdown of expression of endogenous proteins in immune cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Application No.PCT/US2020/026571, filed Apr. 3, 2020 which claims priority from UnitedStates Provisional Application Nos. 62/828,794, filed Apr. 3, 2019,62/843,804, filed May 6, 2019, 62/900,126, filed Sep. 13, 2019,62/930,905, filed Nov. 5, 2019, and 63/000,774, filed Mar. 27, 2020,which applications are hereby incorporated in their entirety byreference in this application.

FIELD OF THE INVENTION

The invention relates to the field of oncology, cancer immunotherapy,molecular biology and recombinant nucleic acid technology. Inparticular, the invention relates to genetically-modified immune cellscomprising a microRNA-adapted shRNA (shRNAmiR) molecule that enablesstable knockdown of a particular target gene. The invention furtherrelates to the use of such genetically-modified immune cells forreducing the expression of an endogenous protein and treating a disease,including cancer, in a subject.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 3, 2020 isnamed PBIO-037WO_Seq_List_4-20, and is 58,911 bytes in size.

BACKGROUND OF THE INVENTION

T cell adoptive immunotherapy is a promising approach for cancertreatment. The immunotherapy treatment methods disclosed herein utilizeisolated human T cells that have been genetically-modified to enhancetheir specificity for a specific tumor associated antigen. Geneticmodification may involve the expression of a chimeric antigen receptoror an exogenous T cell receptor to graft antigen specificity onto the Tcell. By contrast to exogenous T cell receptors, chimeric antigenreceptors derive their specificity from the variable domains of amonoclonal antibody. Thus, T cells expressing chimeric antigen receptors(CAR T cells) induce tumor immunoreactivity in a majorhistocompatibility complex non-restricted manner. T cell adoptiveimmunotherapy has been utilized as a clinical therapy for a number ofcancers, including B cell malignancies (e.g., acute lymphoblasticleukemia, B cell non-Hodgkin lymphoma, acute myeloid leukemia, andchronic lymphocytic leukemia), multiple myeloma, neuroblastoma,glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma,prostate cancer, pancreatic cancer, and others.

Despite its potential usefulness as a cancer treatment, adoptiveimmunotherapy with CAR T cells has been limited, in part, by expressionof the endogenous T cell receptor on the cell surface. CAR T cellsexpressing an endogenous T cell receptor may recognize major and minorhistocompatibility antigens following administration to an allogeneicpatient, which can lead to the development of graft-versus-host-disease(GVHD). As a result, clinical trials have largely focused on the use ofautologous CART cells, wherein a patient's T cells are isolated,genetically-modified to incorporate a chimeric antigen receptor, andthen re-infused into the same patient. An autologous approach providesimmune tolerance to the administered CAR T cells; however, this approachis constrained by both the time and expense necessary to producepatient-specific CAR T cells after a patient's cancer has beendiagnosed.

Thus, it would be advantageous to develop “off the shelf” CAR T cells,prepared using T cells from a third party, healthy donor, that havereduced expression, or have no detectable cell-surface expression, of anendogenous T cell receptor (e.g., an alpha/beta T cell receptor) and donot initiate GVHD upon administration. Such products could be generatedand validated in advance of diagnosis and could be made available topatients as soon as necessary. Therefore, a need exists for thedevelopment of allogeneic CAR T cells that lack an endogenous T cellreceptor in order to prevent the occurrence of GVHD.

To this end, engineered meganucleases having specificity for the beta-2microglobulin gene have been generated in order to fully knockout theexpression of the beta-2 microglobulin (B2M) protein expression (see,for example, International Publication No. WO 2017/112859). B2M is acomponent of the major histocompatibility complex (MHC) class Imolecule, which will not assemble on the cell surface without B2Mpresent. Thus, knockout of B2M is a means for eliminating MHC class Imolecules which should reduce GVHD when CAR T cells are administered toallogeneic patients.

A consequence of fully eliminating B2M and MHC class I moleculeexpression on the cell surface of CAR T cells, however, is that theybecome more susceptible to targeting by natural killer (NK) cells whichsee them as non-self. In view of this phenomenon, a knockdown approachfor CAR T cells was developed in order to produce an incompleteknockdown of B2M (see, for example, International Publication No. WO2018/208837). Essentially, a cassette comprising a B2M-targetedshRNA-coding sequence was introduced into the T cell receptor alphaconstant region gene of the T cell by nuclease-mediated targetedinsertion. The shRNA-coding sequence was included on a cassette thatalso comprised a CAR coding sequence, allowing for the production of CART cells that were TCR-negative, CAR-positive, and had a partialknockdown of cell-surface B2M. The data in this project demonstratedthat these CAR T cells were indeed less susceptible to NK cell killingthan CAR T cells that exhibited a complete knockout of B2M.

As described herein, however, further experiments showed that thecassette comprising the shRNA-coding sequence was not stable, and thatB2M knockdown was transient. Ultimately, the cell removed theshRNA-coding sequence from the genome, causing a return of B2Mexpression. Therefore, a need remains for the production of CAR T cellsthat can maintain a stable knockdown of endogenous proteins, such asB2M. In searching for an answer to this problem in the art, a technologywas discovered herein that can be used to produce genetic knockdown ofvarious degrees of proteins of interest in immune cells.

SUMMARY OF THE INVENTION

The present invention provides genetically-modified immune cells (andpopulations thereof) expressing a microRNA-adapted shRNA (shRNAmiR) thatreduces the expression of a target protein. Using shRNAmiRs for knockingdown the expression of a target protein allows for stable knockdown ofprotein expression, which is ideal for those target proteins for whichknockdown, and not knockout, is preferred. For example, immune cellsthat express beta-2 microglobulin (B2M) at reduced levels via expressionof B2M-targeted shRNAmiRs are less sensitive to cytolysis by naturalkiller (NK) cells than those cells in which B2M expression has beenknocked out by gene inactivation. Thus, further provided are methods forreducing the expression of an endogenous protein in an immune cell byintroducing a template nucleic acid comprising a nucleic acid sequenceencoding a shRNAmiR that is inserted into the cell's genome andexpressed in order to reduce the expression of the endogenous protein.

Thus, in one aspect, the invention provides a genetically-modifiedimmune cell comprising in its genome a nucleic acid sequence encoding amicroRNA-adapted shRNA (shRNAmiR). The shRNAmiR is expressed in thegenetically-modified immune cell and reduces expression of a targetprotein in the genetically-modified immune cell. A reduction in targetprotein expression is mediated by the binding of the shRNAmiR guidesequence to mRNA encoding the target protein.

In some embodiments, the genetically-modified immune cell is agenetically-modified T cell, or a cell derived therefrom. In certainembodiments, the genetically-modified immune cell is agenetically-modified natural killer (NK) cell, or a cell derivedtherefrom. In other embodiments, the genetically-modified immune cell isa genetically-modified B cell, or a cell derived therefrom. In variousembodiments, the genetically-modified immune cell is agenetically-modified monocyte or macrophage, or a cell derivedtherefrom.

In some embodiments, the shRNAmiR comprises, from 5′ to 3′: (a) a 5′ miRscaffold domain; (b) a 5′ miR basal stem domain; (c) a passenger strand;(d) a miR loop domain; (e) a guide strand; (f) a 3′ miR basal stemdomain; and (g) a 3′ miR scaffold domain.

In some embodiments, the miR loop domain is a miR-30a loop domain, amiR-15 loop domain, a miR-16 loop domain, a miR-155 loop domain, amiR-22 loop domain, a miR-103 loop domain, or a miR-107 loop domain. Inparticular embodiments, the miR loop domain is a miR-30a loop domain.

In certain embodiments, the miR-30a loop domain comprises a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 3. In particular embodiments,the miR-30a loop domain comprises a nucleic acid sequence of SEQ ID NO:3.

In some embodiments, the shRNAmiR comprises a microRNA-E (miR-E)scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103 scaffold, ora miR-107 scaffold. In certain embodiments, the shRNAmiR comprises amiR-E scaffold.

In some embodiments, the shRNAmiR comprises a structure wherein: (a) the5′ miR scaffold domain comprises a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 1; (b) the 5′ miR basal stem domain comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 2; (c) the 3′ miRbasal stem domain comprises a nucleic acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, or more, sequence identity toSEQ ID NO: 4; and/or (d) the 3′ miR scaffold domain comprises a nucleicacid sequence having at least 80%, at least 85%, at least 90%, at least95%, or more, sequence identity to SEQ ID NO: 5.

In certain embodiments, the shRNAmiR comprises a structure wherein: (a)the 5′ miR scaffold domain comprises a nucleic acid sequence of SEQ IDNO: 1; (b) the 5′ miR basal stem domain comprises a nucleic acidsequence of SEQ ID NO: 2; (c) the 3′ miR basal stem domain comprises anucleic acid sequence of SEQ ID NO: 4; and (d) the 3′ miR scaffolddomain comprises a nucleic acid sequence of SEQ ID NO: 5.

In some embodiments, the genetically-modified immune cell comprises inits genome a nucleic acid sequence encoding a chimeric antigen receptor(CAR) or an exogenous T cell receptor (TCR), wherein the CAR or theexogenous TCR is expressed by the genetically-modified immune cell.

In some embodiments, the genetically-modified immune cell comprises inits genome a nucleic acid sequence encoding an HLA class Ihistocompatibility antigen, alpha chain E (HLA-E) fusion protein. Insome embodiments, the HLA-E fusion protein comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 66. In some embodiments, theHLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66.

In some embodiments, the nucleic acid sequence encoding the shRNAmiR islocated within a different gene than the nucleic acid sequence encodingthe CAR or the exogenous TCR. In certain embodiments, the nucleic acidsequence encoding the shRNAmiR, or the nucleic acid sequence encodingthe CAR or the exogenous TCR, is located within a TCR alpha gene or aTCR alpha constant region gene. In particular embodiments, the nucleicacid sequence encoding the shRNAmiR, or the nucleic acid sequenceencoding the CAR or the exogenous TCR, is located within a TCR alphaconstant region gene within a sequence comprising SEQ ID NO: 58.

In some embodiments, the nucleic acid sequence encoding the shRNAmiR islocated within the same gene as the nucleic acid sequence encoding theCAR or the exogenous TCR. In certain embodiments, the gene is a TCRalpha gene or TCR alpha constant region gene. In particular embodiments,the nucleic acid sequence encoding the shRNAmiR and the nucleic acidsequence encoding the CAR or the exogenous TCR is located within a TCRalpha constant region gene within a sequence comprising SEQ ID NO: 58.In certain embodiments, the nucleic acid sequence encoding the shRNAmiRand the nucleic acid encoding the CAR or the exogenous TCR are within acassette in the gene. In some such embodiments, the nucleic acidsequence encoding the shRNAmiR and the nucleic acid sequence encodingthe CAR or the exogenous TCR are operably linked to a same promoter. Insome such embodiments, the genetically-modified immune cell comprises inits genome a cassette comprising, from 5′ to 3′: (a) the nucleic acidsequence encoding the CAR or the exogenous TCR; and (b) the nucleic acidsequence encoding the shRNAmiR. In other such embodiments, thegenetically-modified immune cell comprises in its genome a cassettecomprising, from 5′ to 3′: (a) the nucleic acid sequence encoding theshRNAmiR; and (b) the nucleic acid sequence encoding the CAR or theexogenous TCR. In some such embodiments, the nucleic acid sequenceencoding the CAR or the exogenous TCR and the nucleic acid sequenceencoding the shRNAmiR are separated by a 2 A or IRES sequence. Incertain such embodiments, the nucleic acid sequence encoding theshRNAmiR is in the same orientation as the nucleic acid sequenceencoding the CAR or the exogenous TCR. In other such embodiments, thenucleic acid sequence encoding the shRNAmiR is in a reverse orientationas the nucleic acid sequence encoding the CAR or the exogenous TCR. Insome such embodiments, an intron sequence is positioned within thenucleic acid sequence encoding the CAR or the exogenous TCR, and thenucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence. In some such embodiments, the cassette comprises apromoter that is operably linked to the nucleic acid sequence encodingthe shRNAmiR and the nucleic acid sequence encoding the CAR or theexogenous TCR. In some such embodiments, the cassette comprises atermination signal.

In certain embodiments, the nucleic acid sequence encoding the shRNAmiRis located within the same gene as the nucleic acid sequence encodingthe HLA-E fusion protein. In some embodiments, the gene is a TCR alphagene or a TCR alpha constant region gene. In some embodiments, thenucleic acid sequence encoding the shRNAmiR and the nucleic acidsequence encoding the HLA-E fusion protein are within a cassette in thegene. In some such embodiments, the nucleic acid sequence encoding theshRNAmiR and nucleic acid sequence encoding the HLA-E fusion protein areoperably linked to a same promoter. In some such embodiments, thegenetically-modified immune cell comprises in its genome a cassettecomprising, from 5′ to 3′: (a) the nucleic acid sequence encoding theHLA-E fusion protein; and (b) the nucleic acid sequence encoding theshRNAmiR. In some such embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising, from 5′ to 3′: (a) thenucleic acid sequence encoding the shRNAmiR; and (b) the nucleic acidsequence encoding the HLA-E fusion protein. In some such embodiments,the nucleic acid sequence encoding the HLA-E fusion protein and thenucleic acid sequence encoding the shRNAmiR are separated by a 2 A orIRES sequence. In certain such embodiments, an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, wherein the nucleic acid sequence encoding the shRNAmiR ispositioned within the intron sequence. In some such embodiments, thecassette comprises a promoter, wherein the nucleic acid sequenceencoding the shRNAmiR and nucleic acid sequence encoding the HLA-Efusion protein are operably linked to the promoter. In some suchembodiments, the cassette comprises a termination signal.

In some embodiments, the nucleic acid sequence encoding the shRNAmiR,the nucleic acid sequence encoding the CAR or the exogenous TCR, and thenucleic acid sequence encoding the HLA-E fusion protein are locatedwithin the same gene. In some embodiments, the gene is a TCR alpha geneor a TCR alpha constant region gene. In some embodiments, the nucleicacid sequence encoding the shRNAmiR, the nucleic acid sequence encodingthe CAR or the exogenous TCR, and the nucleic acid sequence encoding theHLA-E fusion protein are within a cassette in the gene. In some suchembodiments, the nucleic acid sequence encoding the shRNAmiR, thenucleic acid sequence encoding the CAR or the exogenous TCR, and thenucleic acid sequence encoding the HLA-E fusion protein are operablylinked to a same promoter. In some such embodiments, thegenetically-modified immune cell comprises within its genome a cassettecomprising: (a) the nucleic acid sequence encoding the CAR or theexogenous TCR; (b) a 2 A or IRES sequence; (c) the nucleic acid sequenceencoding the HLA-E fusion protein; and (d) the nucleic acid sequenceencoding the shRNAmiR. In some such embodiments, an intron sequence ispositioned within the nucleic acid sequence encoding the CAR or theexogenous TCR, wherein the nucleic acid sequence encoding the shRNAmiRis positioned within the intron sequence. In other such embodiments, anintron sequence is positioned within the nucleic acid sequence encodingthe HLA-E fusion protein, wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence. In some suchembodiments, the cassette comprises a promoter that is operably linkedto the nucleic acid sequence encoding the CAR or the exogenous TCR, thenucleic acid sequence encoding the HLA-E fusion protein, and the nucleicacid sequence encoding the shRNAmiR. In some such embodiments, thecassette comprises a termination signal.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a promoter;(b) the nucleic acid sequence encoding the CAR or the exogenous TCR; (c)a 2 A or IRES sequence; (d) the nucleic acid sequence encoding the HLA-Efusion protein, wherein an intron sequence is positioned within thenucleic acid sequence encoding the HLA-E fusion protein, and wherein thenucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (e) optionally a termination signal; wherein thenucleic acid sequence encoding the CAR or the exogenous TCR, the nucleicacid sequence encoding the HLA-E fusion protein, and the nucleic acidsequence encoding the shRNAmiR are operably linked to the promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a promoter;(b) the nucleic acid sequence encoding the HLA-E fusion protein, whereinan intron sequence is positioned within the nucleic acid sequenceencoding the HLA-E fusion protein, and wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the CAR orthe exogenous TCR; and (e) optionally a termination signal; wherein thenucleic acid sequence encoding the CAR or the exogenous TCR, the nucleicacid sequence encoding the HLA-E fusion protein, and the nucleic acidsequence encoding the shRNAmiR are operably linked to the promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a promoter;(b) the nucleic acid sequence encoding the CAR or the exogenous TCR,wherein an intron sequence is positioned within the nucleic acidsequence encoding the CAR or the exogenous TCR, and wherein the nucleicacid sequence encoding the shRNAmiR is positioned within the intronsequence; (c) a 2 A or IRES sequence; (d) the nucleic acid sequenceencoding the HLA-E fusion protein; and (e) optionally a terminationsignal; wherein the nucleic acid sequence encoding the CAR or theexogenous TCR, the nucleic acid sequence encoding the HLA-E fusionprotein, and the nucleic acid sequence encoding the shRNAmiR areoperably linked to the promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a promoter;(b) the nucleic acid sequence encoding the HLA-E fusion protein; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the CAR orthe exogenous TCR, wherein an intron sequence is positioned within thenucleic acid sequence encoding the CAR or the exogenous TCR, and whereinthe nucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (e) optionally a termination signal; wherein thenucleic acid sequence encoding the CAR or the exogenous TCR, the nucleicacid sequence encoding the HLA-E fusion protein, and the nucleic acidsequence encoding the shRNAmiR are operably linked to the promoter.

In some embodiments described above, the intron sequence is a syntheticintron sequence. In certain embodiments, the intron sequence comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 69. In particularembodiments, the intron sequence comprises a nucleic acid sequence ofSEQ ID NO: 69.

In some embodiments described above, the termination signal is a polyAsequence or a bovine growth hormone (BGH) termination signal. In certainembodiments, the polyA sequence comprises a nucleic acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 68. In particular embodiments, the polyAsequence comprises a nucleic acid sequence of SEQ ID NO: 68. In certainembodiments, the BGH termination signal comprises a nucleic acidsequence having least 80%, at least 85%, at least 90%, at least 95%, ormore, sequence identity to SEQ ID NO: 71. In particular embodiments, theBGH termination signal comprises a nucleic acid sequence of SEQ ID NO:71.

In some embodiments described above, the promoter comprises a nucleicacid sequence having at least 80%, at least 85%, at least 90%, at least95%, or more, sequence identity to SEQ ID NO: 67. In particularembodiments, the promoter comprises a nucleic acid sequence of SEQ IDNO: 67.

In some embodiments described above, the 2 A sequence is a P2 A/furinsite comprising a nucleic acid sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 70. In particular embodiments, the 2 A sequence is a P2 A/furin sitecomprising a nucleic acid sequence of SEQ ID NO: 70.

In some embodiments described above, the CAR comprises a signal peptidecomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73. Inparticular embodiments, the CAR comprises a signal peptide comprising anamino acid sequence of SEQ ID NO: 73.

In particular embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising, from 5′ to 3′: (a) apromoter comprising a nucleic acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, or more, sequence identity to SEQID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein theCAR comprises a signal peptide comprising an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 73; (c) a P2 A/furin site comprising anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 70; (d) thenucleic acid sequence encoding the HLA-E fusion protein, wherein theHLA-E fusion protein comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 66, and wherein an intron sequence is positionedwithin the nucleic acid sequence encoding the HLA-E fusion protein,wherein the intron sequence comprises a nucleic acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 69, and wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence; and (e)optionally a termination signal comprising a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 68; wherein the nucleic acid sequenceencoding the CAR, the nucleic acid sequence encoding the HLA-E fusionprotein, and the nucleic acid sequence encoding the shRNAmiR areoperably linked to the promoter.

In particular embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising, from 5′ to 3′: (a) apromoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b) thenucleic acid sequence encoding the CAR, wherein the CAR comprises asignal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c) aP2 A/furin site comprising a nucleic acid sequence of SEQ ID NO: 70; (d)the nucleic acid sequence encoding the HLA-E fusion protein, wherein theHLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66,and wherein an intron sequence is positioned within the nucleic acidsequence encoding the HLA-E fusion protein, wherein the intron sequencecomprises a nucleic acid sequence of SEQ ID NO: 69, and wherein thenucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (e) optionally a termination signal comprising anucleic acid sequence of SEQ ID NO: 68; wherein the nucleic acidsequence encoding the CAR, the nucleic acid sequence encoding the HLA-Efusion protein, and the nucleic acid sequence encoding the shRNAmiR areoperably linked to the promoter.

In particular embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 74, wherein the cassette is positionedin the genome within a TCR alpha constant region gene. In particularembodiments, the genetically-modified immune cell comprises in itsgenome a cassette comprising a nucleic acid sequence of SEQ ID NO: 74,wherein the cassette is positioned in the genome within a TCR alphaconstant region gene.

In some embodiments described above, the cassette comprises two or morenucleic acids encoding shRNAmiRs. In certain embodiments, the two ormore nucleic acids can encode the same shRNAmiR. In some embodiments,the two or more nucleic acids can encode different shRNAmiRs that reducethe expression of the same target protein. In other embodiments, the twoor more nucleic acids encode different shRNAmiRs that reduce theexpression of different target proteins. In certain embodiments, thecassette can comprise two or more nucleic acids encoding differentshRNAmiRs described herein. In particular embodiments, the cassette cancomprise a nucleic acid sequence encoding a shRNAmiR that reduces theexpression of B2M, and a nucleic acid sequence encoding a shRNAmiR thatreduces the expression of CD52.

In some embodiments, the nucleic acid sequence encoding the shRNAmiR andthe nucleic acid sequence encoding the CAR or the exogenous TCR arelocated in the same gene and are operably linked to different promoters.In some such embodiments, the genetically-modified immune cell comprisesin its genome a cassette comprising, from 5′ to 3′: (a) a firstpromoter; (b) the nucleic acid sequence encoding the CAR or exogenousTCR which is operably linked to the first promoter; (c) a secondpromoter; and (d) the nucleic acid sequence encoding the shRNAmiR whichis operably linked to the second promoter. In other such embodiments,the genetically-modified immune cell comprises in its genome a cassettecomprising, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the shRNAmiR which is operably linked to the firstpromoter; (c) a second promoter; and (d) the nucleic acid sequenceencoding the CAR or exogenous TCR which is operably linked to the secondpromoter. In some such embodiments, the nucleic acid sequence encodingthe shRNAmiR is in the same orientation as the nucleic acid sequenceencoding the CAR or exogenous TCR. In other such embodiments, thenucleic acid sequence encoding the shRNAmiR is in a reverse orientationas the nucleic acid sequence encoding the CAR or exogenous TCR. Incertain such embodiments, the first promoter and the second promoter areidentical. In other embodiments, the first promoter and the secondpromoter are different. In some such embodiments, the cassette comprisesone or more termination signals.

In some embodiments, the nucleic acid sequence encoding the shRNAmiR andthe nucleic acid sequence encoding the HLA-E fusion protein are operablylinked to different promoters. In some such embodiments, thegenetically-modified immune cell comprises in its genome a cassettecomprising, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the HLA-E fusion protein which is operably linked tothe first promoter; (c) a second promoter; and (d) the nucleic acidsequence encoding the shRNAmiR which is operably linked to the secondpromoter. In other such embodiments, the genetically-modified immunecell comprises in its genome a cassette comprising, from 5′ to 3′: (a) afirst promoter; (b) the nucleic acid sequence encoding the shRNAmiRwhich is operably linked to the first promoter; (c) a second promoter;and (d) the nucleic acid sequence encoding the HLA-E fusion proteinwhich is operably linked to the second promoter. In certain suchembodiments, the first promoter and the second promoter are identical.In other such embodiments, the first promoter and the second promoterare different. In some such embodiments, the cassette comprises one ormore termination signals.

In some embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising: (a) the nucleic acid sequenceencoding the CAR or the exogenous TCR; (b) the nucleic acid sequenceencoding the HLA-E fusion protein; and (c) the nucleic acid sequenceencoding the shRNAmiR; wherein the nucleic acid sequence encoding theCAR or the exogenous TCR is operably linked to a first promoter, andwherein the nucleic acid sequence encoding the HLA-E fusion protein andthe nucleic acid sequence encoding the shRNAmiR are operably linked to asecond promoter. In some such embodiments, an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, wherein the nucleic acid sequence encoding the shRNAmiR ispositioned within the intron sequence. In some embodiments, thegenetically-modified immune cell comprises within its genome a cassettecomprising: (a) the nucleic acid sequence encoding the CAR or theexogenous TCR; (b) the nucleic acid sequence encoding the HLA-E fusionprotein; and (c) the nucleic acid sequence encoding the shRNAmiR;wherein the nucleic acid sequence encoding the CAR or the exogenous TCRand the nucleic acid sequence encoding the shRNAmiR are operably linkedto a first promoter, and wherein the nucleic acid sequence encoding theHLA-E fusion protein is operably linked to a second promoter. In somesuch embodiments, an intron sequence is positioned within the nucleicacid sequence encoding the CAR or the exogenous TCR, wherein the nucleicacid sequence encoding the shRNAmiR is positioned within the intronsequence. In some such embodiments, the cassette comprises a firsttermination signal capable of terminating transcription of the CAR orthe exogenous TCR, and a second termination signal capable ofterminating transcription of the HLA-E fusion protein. In other suchembodiments, the cassette comprises a first termination signal capableof terminating transcription of the HLA-E fusion protein, and a secondtermination signal capable of terminating transcription of the CAR orthe exogenous TCR.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a firstpromoter; (b) the nucleic acid sequence encoding the CAR or theexogenous TCR; (c) optionally a first termination signal; (d) a secondpromoter; (e) the nucleic acid sequence encoding the HLA-E fusionprotein, wherein an intron sequence is positioned within the nucleicacid sequence encoding the HLA-E fusion protein, and wherein the nucleicacid sequence encoding the shRNAmiR is positioned within the intronsequence; and (f) optionally a second termination signal; wherein thenucleic acid sequence encoding the CAR or the exogenous TCR is operablylinked to the first promoter, and wherein the nucleic acid sequenceencoding the HLA-E fusion protein and the nucleic acid sequence encodingthe shRNAmiR are operably linked to the second promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a firstpromoter; (b) the nucleic acid sequence encoding the HLA-E fusionprotein, wherein an intron sequence is positioned within the nucleicacid sequence encoding the HLA-E fusion protein, and wherein the nucleicacid sequence encoding the shRNAmiR is positioned within the intronsequence; (c) optionally a first termination signal; (d) a secondpromoter; (e) the nucleic acid sequence encoding the CAR or theexogenous TCR; and (f) optionally a second termination signal; whereinthe nucleic acid sequence encoding the HLA-E fusion protein and thenucleic acid sequence encoding the shRNAmiR are operably linked to thefirst promoter, and wherein the nucleic acid sequence encoding the CARor the exogenous TCR is operably linked to the second promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a firstpromoter; (b) the nucleic acid sequence encoding the CAR or theexogenous TCR, wherein an intron sequence is positioned within thenucleic acid sequence encoding the CAR or the exogenous TCR, and whereinthe nucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; (c) optionally a first termination signal; (d) a secondpromoter; (e) the nucleic acid sequence encoding the HLA-E fusionprotein; and (f) optionally a second termination signal; wherein thenucleic acid sequence encoding the CAR or the exogenous TCR and thenucleic acid sequence encoding the shRNAmiR are operably linked to thefirst promoter, and wherein the nucleic acid sequence encoding the HLA-Efusion protein is operably linked to the second promoter.

In some such embodiments, the genetically-modified immune cell compriseswithin its genome a cassette comprising, from 5′ to 3′: (a) a firstpromoter; (b) the nucleic acid sequence encoding the HLA-E fusionprotein; (c) optionally a first termination signal; (d) a secondpromoter; (e) the nucleic acid sequence encoding the CAR or theexogenous TCR, wherein an intron sequence is positioned within thenucleic acid sequence encoding the CAR or the exogenous TCR, and whereinthe nucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (f) optionally a second termination signal; whereinthe nucleic acid sequence encoding the HLA-E fusion protein is operablylinked to the first promoter, and wherein the nucleic acid sequenceencoding the CAR or the exogenous TCR and the nucleic acid sequenceencoding the shRNAmiR are operably linked to the second promoter.

In some embodiments described above, the intron sequence is a syntheticintron sequence. In certain embodiments, the intron sequence comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 69. In particularembodiments, the intron sequence comprises a nucleic acid sequence ofSEQ ID NO: 69.

In some embodiments described above, the one or more termination signalsis a polyA sequence or a BGH termination signal.

In some embodiments described above, the first termination signal isidentical to the second termination signal. In some such embodiments,the first termination signal and the second termination signal are apolyA sequence or a BGH termination signal.

In some embodiments described above, the first termination signal isdifferent from the second termination signal. In some embodiments, thefirst termination signal is a polyA sequence and the second terminationsignal is a BGH termination signal. In some embodiments, the firsttermination signal is a BGH termination signal and the secondtermination signal is a polyA sequence.

In some embodiments described above, the polyA sequence comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 68. In certainembodiments, the polyA sequence comprises a nucleic acid sequence of SEQID NO: 68. In some embodiments, the BGH termination signal comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 71. In certainembodiments, the BGH termination signal comprises a nucleic acidsequence of SEQ ID NO: 71.

In some embodiments described above, the first promoter and the secondpromoter are identical. In some such embodiments, the first promoter andthe second promoter are a JeT promoter or an EF1 alpha core promoter.

In some embodiments described above, the first promoter is differentfrom the second promoter. In certain embodiments, the first promoter isa JeT promoter, and the second promoter is an EF1 alpha core promoter.In certain embodiments, the first promoter is an EF1 alpha corepromoter, and the second promoter is a JeT promoter.

In certain embodiments described above, the JeT promoter comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 67. In particularembodiments, the JeT promoter comprises a nucleic acid sequence of SEQID NO: 67. In certain embodiments, the EF1 alpha core promoter comprisesa nucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 72. In someembodiments, the EF1 alpha core promoter comprises a nucleic acidsequence of SEQ ID NO: 72.

In some embodiments described above, the CAR comprises a signal peptidecomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73. Inparticular embodiments, the CAR comprises a signal peptide comprising anamino acid sequence of SEQ ID NO: 73.

In some embodiments, the genetically-modified immune cell comprises inits genome a cassette comprising, from 5′ to 3′: (a) a first promotercomprising a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67;(b) the nucleic acid sequence encoding the CAR, wherein the CARcomprises a signal peptide comprising an amino acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 73; (c) optionally a first termination signalcomprising a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 70;(d) a second promoter comprising a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 72; (e) the nucleic acid sequence encoding theHLA-E fusion protein, wherein the HLA-E fusion protein comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, or more, sequence identity to SEQ ID NO: 66, and wherein anintron sequence is positioned within the nucleic acid sequence encodingthe HLA-E fusion protein, wherein the intron sequence comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 69, and whereinthe nucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (f) optionally a second termination signalcomprising a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71;wherein the nucleic acid sequence encoding the CAR is operably linked tothe first promoter, and wherein the nucleic acid sequence encoding theHLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiRare operably linked to the second promoter.

In particular embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising, from 5′ to 3′: (a) afirst promoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b)the nucleic acid sequence encoding the CAR, wherein the CAR comprises asignal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c)optionally a first termination signal comprising a nucleic acid sequenceof SEQ ID NO: 70; (d) a second promoter comprising a nucleic acidsequence of SEQ ID NO: 72; (e) the nucleic acid sequence encoding theHLA-E fusion protein, wherein the HLA-E fusion protein comprises anamino acid sequence of SEQ ID NO: 66, and wherein an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, wherein the intron sequence comprises a nucleic acid sequenceof SEQ ID NO: 69, and wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence; and (f) optionally asecond termination signal comprising a nucleic acid sequence of SEQ IDNO: 71; wherein the nucleic acid sequence encoding the CAR is operablylinked to the first promoter, and wherein the nucleic acid sequenceencoding the HLA-E fusion protein and the nucleic acid sequence encodingthe shRNAmiR are operably linked to the second promoter.

In particular embodiments, the genetically-modified immune cellcomprises in its genome a cassette comprising a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 75, wherein the cassette is positionedin the genome within a TCR alpha constant region gene. In particularembodiments, the genetically-modified immune cell comprises in itsgenome a cassette comprising a nucleic acid sequence of SEQ ID NO: 75,wherein the cassette is positioned in the genome within a TCR alphaconstant region gene.

In some embodiments described above, the cassette comprises two or morenucleic acids encoding shRNAmiRs. In certain embodiments, the two ormore nucleic acids can encode the same shRNAmiR. In some embodiments,the two or more nucleic acids can encode different shRNAmiRs that reducethe expression of the same target protein. In other embodiments, the twoor more nucleic acids encode different shRNAmiRs that reduce theexpression of different target proteins. In certain embodiments, thecassette can comprise two or more nucleic acids encoding differentshRNAmiRs described herein. In particular embodiments, the cassette cancomprise a nucleic acid sequence encoding a shRNAmiR that reduces theexpression of B2M, and a nucleic acid sequence encoding a shRNAmiR thatreduces the expression of CD52.

In some embodiments, expression of the target protein is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or up to about 99% compared to a control cell.

In some embodiments, the target protein is beta-2 microglobulin, CS1,transforming growth factor-beta receptor 2 (TGFBR2), Cbl proto-oncogeneB (CBL-B), CD52, a TCR alpha gene, a TCR alpha constant region gene,CD7, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), nuclearreceptor subfamily 2 group F member 6 (NR2F6), cytotoxicT-lymphocyte-associated protein 4 (CTLA-4), or C—C chemokine receptortype 5 (CCR5).

In some embodiments, the target protein is beta-2 microglobulin. In somesuch embodiments, cell surface expression of beta-2 microglobulin isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell. In further embodiments, expression of MHC class Imolecules is reduced on the cell surface by at least about 10%, about20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or upto about 99% compared to a control cell. In some such embodiments, thegenetically-modified immune cell has reduced allogenicity compared to acontrol cell.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 18; (b)the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 7and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 8;(c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:9 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:10; (d) the passenger strand comprises a nucleic acid sequence of SEQ IDNO: 11 and the guide strand comprises a nucleic acid sequence of SEQ IDNO: 12; (e) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 13 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 14; or (f) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 15 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 16. In certain such embodiments, the passengerstrand comprises a nucleic acid sequence of SEQ ID NO: 17 and the guidestrand comprises a nucleic acid sequence of SEQ ID NO: 18. In particularsuch embodiments, the nucleic acid sequence encoding the shRNAmiRcomprises a sequence having at least 80%, at least 85%, at least 90%, atleast 95%, or more, sequence identity to SEQ ID NO: 46. In further suchembodiments, the nucleic acid sequence encoding the shRNAmiR comprisesthe sequence of SEQ ID NO: 46.

In some embodiments, the target protein is CS1. In some suchembodiments, cell surface expression of CS1 is reduced by at least about10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or up to about 99% compared to a control cell. In some suchembodiments, the genetically-modified immune cell expresses a CAR havingspecificity for CS1. In further such embodiments, thegenetically-modified immune cell is less susceptible to fratricide by agenetically-modified immune cell expressing a CAR having specificity forCS1 compared to a control cell.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 21 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 22; (b)the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 23and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 24;or (c) the passenger strand comprises a nucleic acid sequence of SEQ IDNO: 25 and the guide strand comprises a nucleic acid sequence of SEQ IDNO: 26. In certain such embodiments, the passenger strand comprises anucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 26. In particular such embodiments,the shRNAmiR comprises a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 50. Infurther such embodiments, the shRNAmiR comprises the sequence of SEQ IDNO: 50.

In some embodiments, the target protein is TGFBR2. In some suchembodiments, the cell surface expression of TGFBR2 is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or up to about 99% compared to a control cell. Infurther embodiments, the genetically-modified immune cell is lesssusceptible to immunosuppression by transforming growth factor B1(TGFB1) compared to a control cell.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 27 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 28; (b)the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 29and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 30;(c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:31 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:32; (d) the passenger strand comprises a nucleic acid sequence of SEQ IDNO: 33 and the guide strand comprises a nucleic acid sequence of SEQ IDNO: 34; or (e) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 35 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 36. In certain such embodiments, the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 31 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 32. In particular suchembodiments, the nucleic acid sequence encoding the shRNAmiR comprises asequence having at least 80%, at least 95%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 53. In further suchembodiments, the nucleic acid sequence encoding the shRNAmiR comprisesthe sequence of SEQ ID NO: 53.

In some embodiments, the target protein is CBL-B. In some suchembodiments, cell surface expression of CBL-B is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell. In furthersuch embodiments, the immune cell is less susceptible to suppression ofT cell receptor (TCR) signaling by degradation of downstream signalingproteins compared to a control cell.

In some embodiments, the target protein is CD52. In some suchembodiments, cell surface expression of CD52 is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell. In furtherembodiments, the genetically-modified immune cell is less susceptible toCD52 antibody-induced cell death.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 38; or(b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:39 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:40. In certain such embodiments, the passenger strand comprises anucleic acid sequence of SEQ ID NO: 37 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 38. In particular such embodiments,the nucleic acid sequence encoding the shRNAmiR comprises a sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 56. In further such embodiments, thenucleic acid sequence encoding the shRNAmiR comprises the sequence ofSEQ ID NO: 56.

In some embodiments, the target protein is DCK. In some suchembodiments, cell surface expression of DCK is reduced by at least about10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or up to about 99% compared to a control cell. In further suchembodiments, the genetically-modified immune cell is less susceptible toeffects of purine nucleoside analogs (e.g., fludarabine) on cellproliferation.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 77; (b)the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 78and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 79;(c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:80 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:81; (d) the passenger strand comprises a nucleic acid sequence of SEQ IDNO: 82 and the guide strand comprises a nucleic acid sequence of SEQ IDNO: 83; or (e) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 84 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 85. In particular such embodiments, the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 76 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 77. In particular suchembodiments, the nucleic acid sequence encoding the shRNAmiR comprises asequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 86. In further suchembodiments, the nucleic acid sequence encoding the shRNAmiR comprisesthe sequence of SEQ ID NO: 86.

In some embodiments, the target protein is GR. In some such embodiments,cell surface expression of GR is reduced by at least about 10%, about20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or upto about 99% compared to a control cell. In further such embodiments,the genetically-modified immune cell is less susceptible to effects ofglucocorticoids (e.g., dexamethasone), such as reduced proliferation.

In some such embodiments, the shRNAmiR has a structure wherein: (a) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 91 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 92; (b)the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 93and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 94;(c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:95 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:96; (d) the passenger strand comprises a nucleic acid sequence of SEQ IDNO: 97 and the guide strand comprises a nucleic acid sequence of SEQ IDNO: 98; (e) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 99 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 100; (f) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 101 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 102; (g) the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 103 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 104; (h) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 105 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 106; or (i) the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 107 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 108. In particular suchembodiments, the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 95 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 96. In particular such embodiments, the nucleic acid sequenceencoding the shRNAmiR comprises a sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 111. In further such embodiments, the nucleic acid sequence encodingthe shRNAmiR comprises the sequence of SEQ ID NO: 111.

In another aspect, the invention provides a method for reducing theexpression of an endogenous protein in an immune cell, the methodcomprising introducing into the immune cell a template nucleic acidcomprising a nucleic acid sequence encoding a shRNAmiR, wherein thetemplate nucleic acid is inserted into the genome of the immune cell.The shRNAmiR is expressed in the immune cell and reduces expression ofan endogenous target protein in the immune cell. A reduction in targetprotein expression is mediated by the binding of the shRNAmiR guidesequence to mRNA encoding the target protein.

In some embodiments of the method, the immune cell is a T cell, or acell derived therefrom. In certain embodiments, the immune cell is anatural killer (NK) cell, or a cell derived therefrom. In otherembodiments, the immune cell is a B cell, or a cell derived therefrom.In various embodiments, the immune cell is a monocyte or macrophage, ora cell derived therefrom.

In some embodiments of the method, the template nucleic acid is insertedinto the genome of the immune cell by random integration. In some suchembodiments of the method, the template nucleic acid is introduced intothe immune cell using a viral vector (i.e., a recombinant virus), suchas a lentiviral vector (i.e., a recombinant lentivirus).

In some embodiments of the method, the immune cell expresses a CAR orexogenous TCR.

In some embodiments, the method further comprises introducing into theimmune cell a second nucleic acid encoding an engineered nuclease havingspecificity for a recognition sequence in the genome of the immune cell.The engineered nuclease is expressed in the immune cell and generates acleavage site at the recognition sequence. The template nucleic acidcomprising a nucleic acid sequence encoding the shRNAmiR is insertedinto the genome of the immune cell at the cleavage site. In some suchembodiments of the method, the template nucleic acid is flanked byhomology arms having homology to sequences flanking the recognitionsequence, and the template nucleic acid is inserted at the cleavage siteby homologous recombination. In some such embodiments of the method, thetemplate nucleic acid is introduced into the immune cell using a viralvector (i.e., a recombinant virus). In some such embodiments, the viralvector is a recombinant AAV vector (i.e., a recombinant AAV). Inparticular embodiments, the AAV vector has a serotype of AAV2 or AAV6.In some such embodiments of the method, the recognition sequence iswithin a target gene. In some such embodiments of the method, theexpression of a protein encoded by the target gene is disrupted in theimmune cell. In certain such embodiments of the method, the target geneis a TCR alpha gene or a TCR alpha constant region gene, and the immunecell does not have detectable cell-surface expression of an endogenousTCR (e.g., an alpha/beta TCR). In some such embodiments of the method,the engineered nuclease is an engineered meganuclease, a zinc fingernuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or amegaTAL. In particular such embodiments of the method, the engineerednuclease is an engineered meganuclease. In certain such embodiments ofthe method, the second nucleic acid encoding the engineered nuclease isintroduced using an mRNA.

In some embodiments of the method, the immune cell into which thetemplate nucleic acid is introduced further comprises in its genome anucleic acid sequence encoding a CAR or exogenous TCR. In certainembodiments of the method, the immune cell into which the templatenucleic acid is introduced further comprises in its genome a nucleicacid sequence encoding an HLA-E fusion protein.

In some embodiments of the method, the template nucleic acid furthercomprises a nucleic acid sequence encoding a CAR or an exogenous TCR,wherein the CAR or the exogenous TCR is expressed by the immune cell. Insome such embodiments, the nucleic acid sequence encoding the shRNAmiRand the nucleic acid sequence encoding the CAR or the exogenous TCR areoperably linked to a same promoter in the immune cell followingintroduction of the template nucleic acid at the cleavage site. In somesuch embodiments of the method, the template nucleic acid comprises,from 5′ to 3′: (a) the nucleic acid sequence encoding the CAR or theexogenous TCR; and (b) the nucleic acid sequence encoding the shRNAmiR.In other such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) the nucleic acid sequence encoding theshRNAmiR; and (b) the nucleic acid sequence encoding the CAR or theexogenous TCR. In certain such embodiments of the method, the nucleicacid sequence encoding the CAR or the exogenous TCR and the nucleic acidsequence encoding the shRNAmiR are separated by a 2 A or IRES sequence.In some such embodiments of the method, the nucleic acid sequenceencoding the shRNAmiR is in the same orientation as the nucleic acidsequence encoding the CAR or the exogenous TCR. In other suchembodiments of the method, the nucleic acid sequence encoding theshRNAmiR is in a reverse orientation as the nucleic acid sequenceencoding the CAR or the exogenous TCR. In some such embodiments of themethod, an intron sequence is positioned within the nucleic acidsequence encoding the CAR or the exogenous TCR, wherein the nucleic acidsequence encoding the shRNAmiR is positioned within the intron sequence.In certain such embodiments of the method, the template nucleic acidcomprises a promoter, wherein the promoter is operably linked to thenucleic acid sequence encoding the CAR or the exogenous TCR and to thenucleic acid sequence encoding the shRNAmiR. In some such embodiments ofthe method, the template nucleic acid comprises a termination signal.

In some embodiments of the method, the template nucleic acid comprises anucleic acid sequence encoding an HLA-E fusion protein, wherein theHLA-E fusion protein is expressed by the immune cell. In some suchembodiments of the method, the shRNAmiR and the nucleic acid sequenceencoding the HLA-E fusion protein are operably linked to a same promoterin the immune cell following introduction of the template nucleic acidat the cleavage site. In certain such embodiments of the method, thetemplate nucleic acid comprises, from 5′ to 3′: (a) the nucleic acidsequence encoding the HLA-E fusion protein; and (b) the nucleic acidsequence encoding the shRNAmiR. In other such embodiments of the method,the template nucleic acid comprises, from 5′ to 3′: (a) the nucleic acidsequence encoding the shRNAmiR; and (b) the nucleic acid sequenceencoding the HLA-E fusion protein. In some such embodiments of themethod, the nucleic acid sequence encoding the HLA-E fusion protein andthe nucleic acid sequence encoding the shRNAmiR are separated by a 2 Aor IRES sequence. In certain such embodiments, of the method, an intronsequence is positioned within the nucleic acid sequence encoding theHLA-E fusion protein, wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence. In some suchembodiments of the method, the template nucleic acid comprises apromoter, wherein the promoter is operably linked to the nucleic acidsequence encoding the HLA-E fusion protein and to the nucleic acidsequence encoding the shRNAmiR. In certain such embodiments of themethod, the template nucleic acid comprises a termination signal.

In some embodiments of the method, the template nucleic acid comprises anucleic acid sequence encoding a CAR or an exogenous TCR and a nucleicacid sequence encoding an HLA-E fusion protein, wherein the CAR or theexogenous TCR and the HLA-E fusion protein are expressed by the immunecell. In some embodiments of the method, the nucleic acid sequenceencoding the shRNAmiR, the nucleic acid sequence encoding the CAR or theexogenous TCR, and the nucleic acid sequence encoding the HLA-E fusionprotein are operably linked to a same promoter following introduction ofthe template nucleic acid at the cleavage site. In some such embodimentsof the method, the template nucleic acid comprises: (a) the nucleic acidsequence encoding the CAR or the exogenous TCR; (b) a 2 A or IRESsequence; (c) the nucleic acid sequence encoding the HLA-E fusionprotein; and (d) the nucleic acid sequence encoding the shRNAmiR. Incertain such embodiments of the method, an intron sequence is positionedwithin the nucleic acid sequence encoding the HLA-E fusion protein, andwherein the nucleic acid sequence encoding the shRNAmiR is positionedwithin the intron sequence. In other such embodiments of the method, anintron sequence is positioned within the nucleic acid sequence encodingthe CAR or the exogenous TCR, and wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence. In somesuch embodiments of the method, the template nucleic acid comprises apromoter that is operably linked to the nucleic acid sequence encodingthe CAR or the exogenous TCR, the nucleic acid sequence encoding theHLA-E fusion protein, and the nucleic acid sequence encoding theshRNAmiR. In certain such embodiments of the method, the templatenucleic acid comprises a termination signal.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter; (b) the nucleic acid sequenceencoding the CAR or the exogenous TCR; (c) a 2 A or IRES sequence; (d)the nucleic acid sequence encoding the HLA-E fusion protein, wherein anintron sequence is positioned within the nucleic acid sequence encodingthe HLA-E fusion protein, and wherein the nucleic acid sequence encodingthe shRNAmiR is positioned within the intron sequence; and (e)optionally a termination signal; wherein the nucleic acid sequenceencoding the CAR or the exogenous TCR, the nucleic acid sequenceencoding the HLA-E fusion protein, and the nucleic acid sequenceencoding the shRNAmiR are operably linked to the promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter; (b) the nucleic acid sequenceencoding the HLA-E fusion protein, wherein an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, and wherein the nucleic acid sequence encoding the shRNAmiR ispositioned within the intron sequence; (c) a 2 A or IRES sequence; (d)the nucleic acid sequence encoding the CAR or the exogenous TCR; and (e)optionally a termination signal; wherein the nucleic acid sequenceencoding the CAR or the exogenous TCR, the nucleic acid sequenceencoding the HLA-E fusion protein, and the nucleic acid sequenceencoding the shRNAmiR are operably linked to the promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter; (b) the nucleic acid sequenceencoding the CAR or the exogenous TCR, wherein an intron sequence ispositioned within the nucleic acid sequence encoding the CAR or theexogenous TCR, and wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence; (c) a 2 A or IRESsequence; (d) the nucleic acid sequence encoding the HLA-E fusionprotein; and (e) optionally a termination signal; wherein the nucleicacid sequence encoding the CAR or the exogenous TCR, the nucleic acidsequence encoding the HLA-E fusion protein, and the nucleic acidsequence encoding the shRNAmiR are operably linked to the promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter; (b) the nucleic acid sequenceencoding the HLA-E fusion protein; (c) a 2 A or IRES sequence; (d) thenucleic acid sequence encoding the CAR or the exogenous TCR, wherein anintron sequence is positioned within the nucleic acid sequence encodingthe CAR or the exogenous TCR, and wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence; and (e)optionally a termination signal; wherein the nucleic acid sequenceencoding the CAR or the exogenous TCR, the nucleic acid sequenceencoding the HLA-E fusion protein, and the nucleic acid sequenceencoding the shRNAmiR are operably linked to the promoter.

In some embodiments of the method described above, the intron sequenceis a synthetic intron sequence. In certain embodiments of the method,the intron sequence comprises a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 69. In particular embodiments of the method, theintron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

In some embodiments of the method described above, the terminationsignal is a polyA sequence or a bovine growth hormone (BGH) terminationsignal. In certain embodiments of the method, the polyA sequencecomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. Inparticular embodiments of the method, the polyA sequence comprises anucleic acid sequence of SEQ ID NO: 68. In certain embodiments of themethod, the BGH termination signal comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 71. In particular embodiments of themethod, the BGH termination signal comprises a nucleic acid sequence ofSEQ ID NO: 71.

In certain embodiments of the method described above, the promotercomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67. Inparticular embodiments of the method, the promoter comprises a nucleicacid sequence of SEQ ID NO: 67.

In certain embodiments of the method described above, the 2 A sequenceis a P2 A/furin site comprising a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 70. In particular embodiments of the method, the2 A sequence is a P2 A/furin site comprising a nucleic acid sequence ofSEQ ID NO: 70.

In certain embodiments of the method described above, the HLA-E fusionprotein comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 66. In particular embodiments of the method, the HLA-E fusionprotein comprises an amino acid sequence of SEQ ID NO: 66.

In certain embodiments of the method described above, the CAR comprisesa signal peptide comprising an amino acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, or more, sequence identity toSEQ ID NO: 73. In particular embodiments of the method, the CARcomprises a signal peptide comprising an amino acid sequence of SEQ IDNO: 73.

In particular embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter comprising a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acidsequence encoding the CAR, wherein the CAR comprises a signal peptidecomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73;(c) a P2 A/furin site comprising a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 70; (d) the nucleic acid sequence encoding theHLA-E fusion protein, wherein the HLA-E fusion protein comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, or more, sequence identity to SEQ ID NO: 66, and wherein anintron sequence is positioned within the nucleic acid sequence encodingthe HLA-E fusion protein, wherein the intron sequence comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 69, and whereinthe nucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (e) optionally a termination signal comprising anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 68; wherein thenucleic acid sequence encoding the CAR, the nucleic acid sequenceencoding the HLA-E fusion protein, and the nucleic acid sequenceencoding the shRNAmiR are operably linked to the promoter.

In particular embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a promoter comprising a nucleic acidsequence of SEQ ID NO: 67; (b) the nucleic acid sequence encoding theCAR, wherein the CAR comprises a signal peptide comprising an amino acidsequence of SEQ ID NO: 73; (c) a P2 A/furin site comprising a nucleicacid sequence of SEQ ID NO: 70; (d) the nucleic acid sequence encodingthe HLA-E fusion protein, wherein the HLA-E fusion protein comprises anamino acid sequence of SEQ ID NO: 66, and wherein an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, wherein the intron sequence comprises a nucleic acid sequenceof SEQ ID NO: 69, and wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence; and (e) optionally atermination signal comprising a nucleic acid sequence of SEQ ID NO: 68;wherein the nucleic acid sequence encoding the CAR, the nucleic acidsequence encoding the HLA-E fusion protein, and the nucleic acidsequence encoding the shRNAmiR are operably linked to the promoter.

In particular embodiments of the method, the template nucleic acidcomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 74,wherein the template nucleic acid is inserted in the genome within a TCRalpha constant region gene. In particular embodiments of the method, thetemplate nucleic acid comprises a nucleic acid sequence of SEQ ID NO:74, wherein the template nucleic acid is inserted in the genome within aTCR alpha constant region gene.

In some embodiments of the method described above, the template nucleicacid comprises two or more nucleic acids encoding shRNAmiRs. In certainembodiments of the method, the two or more nucleic acids can encode thesame shRNAmiR. In some embodiments of the method, the two or morenucleic acids can encode different shRNAmiRs that reduce the expressionof the same target protein. In other embodiments of the method, the twoor more nucleic acids encode different shRNAmiRs that reduce theexpression of different target proteins. In certain embodiments of themethod, the template nucleic acid can comprise two or more nucleic acidsencoding different shRNAmiRs described herein. In particular embodimentsof the method, the template nucleic acid can comprise a nucleic acidsequence encoding a shRNAmiR that reduces the expression of B2M, and anucleic acid sequence encoding a shRNAmiR that reduces the expression ofCD52.

In some embodiments of the method, the nucleic acid sequence encodingthe shRNAmiR and the nucleic acid sequence encoding the CAR or theexogenous TCR are operably linked to different promoters in the immunecell following introduction of the template nucleic acid at the cleavagesite. In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the CAR or exogenous TCR which is operably linked tothe first promoter; (c) a second promoter; and (d) the nucleic acidsequence encoding the shRNAmiR which is operably linked to the secondpromoter. In other such embodiments of the method, the template nucleicacid comprises, from 5′ to 3′: (a) a first promoter; (b) the nucleicacid sequence encoding the shRNAmiR which is operably linked to thefirst promoter; (c) a second promoter; and (d) the nucleic acid sequenceencoding the CAR or exogenous TCR which is operably linked to the secondpromoter. In certain such embodiments of the method, the nucleic acidsequence encoding the shRNAmiR is in the same orientation as the nucleicacid sequence encoding the CAR or exogenous TCR. In other suchembodiments of the method, the nucleic acid sequence encoding theshRNAmiR is in a reverse orientation as the nucleic acid sequenceencoding the CAR or exogenous TCR. In particular such embodiments of themethod, the first promoter and the second promoter are identical. Inother such embodiments of the method, the first promoter and the secondpromoter are different. In some such embodiments of the method, thetemplate nucleic acid comprises one or more termination signals.

In some embodiments of the method, the nucleic acid sequence encodingthe shRNAmiR and the nucleic acid sequence encoding the HLA-E fusionprotein are operably linked to different promoters in the immune cellfollowing introduction of the template nucleic acid at the cleavagesite. In certain such embodiments of the method, the template nucleicacid comprises, from 5′ to 3′: (a) a first promoter; (b) the nucleicacid sequence encoding the HLA-E fusion protein which is operably linkedto the first promoter; (c) a second promoter; and (d) the nucleic acidsequence encoding the shRNAmiR which is operably linked to the secondpromoter. In some such embodiments of the method, the template nucleicacid comprises, from 5′ to 3′: (a) a first promoter; (b) the nucleicacid sequence encoding the shRNAmiR which is operably linked to thefirst promoter; (c) a second promoter; and (d) the nucleic acid sequenceencoding the HLA-E fusion protein which is operably linked to the secondpromoter. In certain such embodiments of the method, the first promoterand the second promoter are identical. In other such embodiments of themethod, the first promoter and the second promoter are different. Insome such embodiments of the method, the template nucleic acid comprisesone or more termination signals.

In some embodiments of the method, the template nucleic acid comprises:(a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b)the nucleic acid sequence encoding the HLA-E fusion protein; and (c) thenucleic acid sequence encoding the shRNAmiR; wherein the nucleic acidsequence encoding the CAR or the exogenous TCR is operably linked to afirst promoter, and wherein the nucleic acid sequence encoding the HLA-Efusion protein and the nucleic acid sequence encoding the shRNAmiR areoperably linked to a second promoter. In some embodiments of the method,an intron sequence is positioned within the nucleic acid sequenceencoding the HLA-E fusion protein, wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence. In someembodiments of the method, the template nucleic acid comprises: (a) thenucleic acid sequence encoding the CAR or the exogenous TCR; (b) thenucleic acid sequence encoding the HLA-E fusion protein; and (c) thenucleic acid sequence encoding the shRNAmiR; wherein the nucleic acidsequence encoding the CAR or the exogenous TCR and the nucleic acidsequence encoding the shRNAmiR are operably linked to a first promoter,and wherein the nucleic acid sequence encoding the HLA-E fusion proteinis operably linked to a second promoter. In some embodiments of themethod, an intron sequence is positioned within the nucleic acidsequence encoding the CAR or the exogenous TCR, wherein the nucleic acidsequence encoding the shRNAmiR is positioned within the intron sequence.

In some such embodiments of the method, the template nucleic acidcomprises a first termination signal capable of terminatingtranscription of the CAR or the exogenous TCR, and a second terminationsignal capable of terminating transcription of the HLA-E fusion protein.In some such embodiments of the method, the template nucleic acidcomprises a first termination signal capable of terminatingtranscription of the HLA-E fusion protein, and a second terminationsignal capable of terminating transcription of the CAR or the exogenousTCR.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the CAR or the exogenous TCR; (c) optionally a firsttermination signal; (d) a second promoter; (e) the nucleic acid sequenceencoding the HLA-E fusion protein, wherein an intron sequence ispositioned within the nucleic acid sequence encoding the HLA-E fusionprotein, and wherein the nucleic acid sequence encoding the shRNAmiR ispositioned within the intron sequence; and (f) optionally a secondtermination signal; wherein the nucleic acid sequence encoding the CARor the exogenous TCR is operably linked to the first promoter, andwherein the nucleic acid sequence encoding the HLA-E fusion protein andthe nucleic acid sequence encoding the shRNAmiR are operably linked tothe second promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the HLA-E fusion protein, wherein an intron sequenceis positioned within the nucleic acid sequence encoding the HLA-E fusionprotein, and wherein the nucleic acid sequence encoding the shRNAmiR ispositioned within the intron sequence; (c) optionally a firsttermination signal; (d) a second promoter; (e) the nucleic acid sequenceencoding the CAR or the exogenous TCR; and (f) optionally a secondtermination signal; wherein the nucleic acid sequence encoding the HLA-Efusion protein and the nucleic acid sequence encoding the shRNAmiR areoperably linked to the first promoter, and wherein the nucleic acidsequence encoding the CAR or the exogenous TCR is operably linked to thesecond promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the CAR or the exogenous TCR, wherein an intronsequence is positioned within the nucleic acid sequence encoding the CARor the exogenous TCR, and wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence; (c) optionally afirst termination signal; (d) a second promoter; (e) the nucleic acidsequence encoding the HLA-E fusion protein; and (f) optionally a secondtermination signal; wherein the nucleic acid sequence encoding the CARor the exogenous TCR and the nucleic acid sequence encoding the shRNAmiRare operably linked to the first promoter, and wherein the nucleic acidsequence encoding the HLA-E fusion protein is operably linked to thesecond promoter.

In some such embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter; (b) the nucleic acidsequence encoding the HLA-E fusion protein; (c) optionally a firsttermination signal; (d) a second promoter; (e) the nucleic acid sequenceencoding the CAR or the exogenous TCR, wherein an intron sequence ispositioned within the nucleic acid sequence encoding the CAR or theexogenous TCR, and wherein the nucleic acid sequence encoding theshRNAmiR is positioned within the intron sequence; and (f) optionally asecond termination signal; wherein the nucleic acid sequence encodingthe HLA-E fusion protein is operably linked to the first promoter, andwherein the nucleic acid sequence encoding the CAR or the exogenous TCRand the nucleic acid sequence encoding the shRNAmiR are operably linkedto the second promoter.

In some embodiments of the method described above, the intron sequenceis a synthetic intron sequence. In certain embodiments of the method,the intron sequence comprises a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 69. In particular embodiments of the method, theintron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

In some embodiments of the method described above, the one or moretermination signals are a polyA sequence or a BGH termination signal.

In some embodiments of the method described above, the first terminationsignal is identical to the second termination signal. In otherembodiments of the method, the first termination signal is differentfrom the second termination signal. In certain embodiments of themethod, the first termination signal is a polyA sequence and the secondtermination signal is a BGH termination signal. In certain embodimentsof the method, the first termination signal is a BGH termination signaland the second termination signal is a polyA sequence.

In certain embodiments of the method described above, the polyA sequencecomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. Inparticular embodiments of the method, the polyA sequence comprises anucleic acid sequence of SEQ ID NO: 68. In certain embodiments of themethod, the BGH termination signal comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 71. In particular embodiments of themethod, the BGH termination signal comprises a nucleic acid sequence ofSEQ ID NO: 71.

In some embodiments of the method described above, the first promoterand the second promoter are identical. In other embodiments of themethod, the first promoter is different from the second promoter. Incertain embodiments of the method, the first promoter is a JeT promoter,and the second promoter is an EF1 alpha core promoter. In certainembodiments of the method, the first promoter is an EF1 alpha corepromoter, and the second promoter is a JeT promoter.

In certain embodiments of the method described above, the JeT promotercomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67. Inparticular embodiments of the method, the JeT promoter comprises anucleic acid sequence of SEQ ID NO: 67. In certain embodiments of themethod, the EF1 alpha core promoter comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 72. In particular embodiments, the EF1alpha core promoter comprises a nucleic acid sequence of SEQ ID NO: 72.

In some embodiments of the method described above, the HLA-E fusionprotein comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 66. In particular embodiments of the method, the HLA-E fusionprotein comprises an amino acid sequence of SEQ ID NO: 66.

In some embodiments of the method described above, the CAR comprises asignal peptide comprising an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, or more, sequence identity to SEQID NO: 73. In particular embodiments of the method, the CAR comprises asignal peptide comprising an amino acid sequence of SEQ ID NO: 73.

In particular embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter comprising a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acidsequence encoding the CAR, wherein the CAR comprises a signal peptidecomprising an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73;(c) optionally a first termination signal comprising a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 70; (d) a second promotercomprising a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 72;(e) the nucleic acid sequence encoding the HLA-E fusion protein, whereinthe HLA-E fusion protein comprises an amino acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 66, and wherein an intron sequence is positionedwithin the nucleic acid sequence encoding the HLA-E fusion protein,wherein the intron sequence comprises a nucleic acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, or more, sequenceidentity to SEQ ID NO: 69, and wherein the nucleic acid sequenceencoding the shRNAmiR is positioned within the intron sequence; and (f)optionally a second termination signal comprising a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 71; wherein the nucleic acidsequence encoding the CAR is operably linked to the first promoter, andwherein the nucleic acid sequence encoding the HLA-E fusion protein andthe nucleic acid sequence encoding the shRNAmiR are operably linked tothe second promoter.

In particular embodiments of the method, the template nucleic acidcomprises, from 5′ to 3′: (a) a first promoter comprising a nucleic acidsequence of SEQ ID NO: 67; (b) the nucleic acid sequence encoding theCAR, wherein the CAR comprises a signal peptide comprising an amino acidsequence of SEQ ID NO: 73; (c) optionally a first termination signalcomprising a nucleic acid sequence of SEQ ID NO: 70; (d) a secondpromoter comprising a nucleic acid sequence of SEQ ID NO: 72; (e) thenucleic acid sequence encoding the HLA-E fusion protein, wherein theHLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66,and wherein an intron sequence is positioned within the nucleic acidsequence encoding the HLA-E fusion protein, wherein the intron sequencecomprises a nucleic acid sequence of SEQ ID NO: 69, and wherein thenucleic acid sequence encoding the shRNAmiR is positioned within theintron sequence; and (f) optionally a second termination signalcomprising a nucleic acid sequence of SEQ ID NO: 71; wherein the nucleicacid sequence encoding the CAR is operably linked to the first promoter,and wherein the nucleic acid sequence encoding the HLA-E fusion proteinand the nucleic acid sequence encoding the shRNAmiR are operably linkedto the second promoter.

In particular embodiments of the method, the template nucleic acidcomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 75,wherein the cassette is inserted in the genome within a TCR alphaconstant region gene. In particular embodiments of the method, thetemplate nucleic acid comprises a nucleic acid sequence of SEQ ID NO:75, wherein the cassette is inserted in the genome within a TCR alphaconstant region gene.

In some embodiments of the method described above, the template nucleicacid comprises two or more nucleic acids encoding shRNAmiRs. In certainembodiments of the method, the two or more nucleic acids can encode thesame shRNAmiR. In some embodiments of the method, the two or morenucleic acids can encode different shRNAmiRs that reduce the expressionof the same target protein. In other embodiments of the method, the twoor more nucleic acids encode different shRNAmiRs that reduce theexpression of different target proteins. In certain embodiments of themethod, the template nucleic acid can comprise two or more nucleic acidsencoding different shRNAmiRs described herein. In particular embodimentsof the method, the template nucleic acid can comprise a nucleic acidsequence encoding a shRNAmiR that reduces the expression of B2M, and anucleic acid sequence encoding a shRNAmiR that reduces the expression ofCD52.

In some embodiments of the method, the shRNAmiR comprises, from 5′ to3′: (a) a 5′ miR scaffold domain; (b) a 5′ miR basal stem domain; (c) apassenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3′miR basal stem domain; and (g) a 3′ miR scaffold domain.

In some embodiments of the method, the miR loop domain is a miR-30a loopdomain, a miR-15 loop domain, a miR-16 loop domain, a miR-155 loopdomain, a miR-22 loop domain, a miR-103 loop domain, or a miR-107 loopdomain. In particular embodiments of the method, the miR loop domain isa miR-30a loop domain.

In certain embodiments of the method, the miR-30a loop domain comprisesa nucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 3. In particularembodiments of the method, the miR-30a loop domain comprises a nucleicacid sequence of SEQ ID NO: 3.

In some embodiments of the method, the shRNAmiR comprises a microRNA-E(miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold,a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103scaffold, or a miR-107 scaffold. In certain embodiments of the method,the shRNAmiR comprises a miR-E scaffold.

In some embodiments of the method, the shRNAmiR comprises a structurewherein: (a) the 5′ miR scaffold domain comprises a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 1; (b) the 5′ miR basal stemdomain comprises a nucleic acid sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 2; (c) the 3′ miR basal stem domain comprises a nucleic acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 4; and/or (d) the 3′ miRscaffold domain comprises a nucleic acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, or more, sequence identity toSEQ ID NO: 5.

In certain embodiments of the method, the shRNAmiR comprises a structurewherein: (a) the 5′ miR scaffold domain comprises a nucleic acidsequence of SEQ ID NO: 1; (b) the 5′ miR basal stem domain comprises anucleic acid sequence of SEQ ID NO: 2; (c) the 3′ miR basal stem domaincomprises a nucleic acid sequence of SEQ ID NO: 4; and (d) the 3′ miRscaffold domain comprises a nucleic acid sequence of SEQ ID NO: 5.

In some embodiments of the method, expression of the target protein isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell.

In some embodiments of the method, the target protein is beta-2microglobulin, CS1, transforming growth factor-beta receptor 2 (TGFBR2),Cbl proto-oncogene B (CBL-B), CD52, a TCR alpha gene, a TCR alphaconstant region gene, CD7, glucocorticoid receptor (GR), deoxycytidinekinase (DCK), nuclear receptor subfamily 2 group F member 6 (NR2F6),cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or C—C chemokinereceptor type 5 (CCR5).

In some embodiments of the method, the target protein is beta-2microglobulin. In some such embodiments of the method, cell surfaceexpression of beta-2 microglobulin is reduced by at least about 10%,about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or up to about 99% compared to a control cell. In further suchembodiments of the method, expression of MHC class I molecules isreduced on the cell surface by at least about 10%, about 20%, about 30%,about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or up to about 99%compared to a control cell. In some such embodiments of the method, theimmune cell has reduced allogenicity compared to a control cell.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 17 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 18; (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 7 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 8; (c) the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 9 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 10; (d) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 11 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 12; (e) the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 13 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 14; or (f) the passengerstrand comprises a nucleic acid sequence of SEQ ID NO: 15 and the guidestrand comprises a nucleic acid sequence of SEQ ID NO: 16. In certainsuch embodiments of the method, the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 17 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 18. In particular such embodiments of themethod, the nucleic acid sequence encoding the shRNAmiR comprises asequence having at least 80%, at least 85%, at least 90%, at least 95%,or more, sequence identity to SEQ ID NO: 46. In further such embodimentsof the method, the nucleic acid sequence encoding the shRNAmiR comprisesthe sequence of SEQ ID NO: 46.

In some embodiments of the method, the target protein is CS1. In somesuch embodiments of the method, cell surface expression of CS1 isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell. In some such embodiments of the method, the immune cellexpresses a CAR having specificity for CS1. In further such embodimentsof the method, the immune cell is less susceptible to fratricide by aimmune cell expressing a CAR having specificity for CS1 compared to acontrol cell.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 21 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 22; (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 23 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 24; or (c) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 26. In certain such embodiments ofthe method, the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 25 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 26. In particular such embodiments of the method, theshRNAmiR comprises a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or more, sequence identity to SEQ ID NO: 50. Infurther such embodiments of the method, the shRNAmiR comprises thesequence of SEQ ID NO: 50.

In some embodiments of the method, the target protein is TGFBR2. In somesuch embodiments of the method, the cell surface expression of TGFBR2 isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell. In further such embodiments of the method, the immune cellis less susceptible to immunosuppression by transforming growth factorB1 (TGFB1) compared to a control cell.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 27 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 28; (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 29 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 30; (c) the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 31 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 32; (d) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 33 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 34; or (e) the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 35 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 36. In certain suchembodiments of the method, the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 31 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 32. In particular such embodiments of the method,the nucleic acid sequence encoding the shRNAmiR comprises a sequencehaving at least 80%, at least 95%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 53. In further such embodiments of themethod, the nucleic acid sequence encoding the shRNAmiR comprises thesequence of SEQ ID NO: 53.

In some embodiments of the method, the target protein is CBL-B. In somesuch embodiments of the method, cell surface expression of CBL-B isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell. In further such embodiments of the method, the immune cellis less susceptible to suppression of T cell receptor (TCR) signaling bydegradation of downstream signaling proteins compared to a control cell.

In some embodiments of the method, the target protein is CD52. In somesuch embodiments of the method, cell surface expression of CD52 isreduced by at least about 10%, about 20%, about 30%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell. In further such embodiments of the method, the immune cellis less susceptible to CD52 antibody-induced cell death.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 37 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 38; or (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 39 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 40. In certain such embodiments of the method,the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 38.In particular such embodiments of the method, the nucleic acid sequenceencoding the shRNAmiR comprises a sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 56. In further such embodiments of the method, the nucleic acidsequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 56.

In some embodiments of the method, the target protein is DCK. In somesuch embodiments, cell surface expression of DCK is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell. In furthersuch embodiments of the method, the immune cell is less susceptible toeffects of purine nucleoside analogs (e.g., fludarabine) on cellproliferation.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 76 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 77; (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 78 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 79; (c) the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 80 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 81; (d) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 82 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 83; or (e) the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 84 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 85. In particular suchembodiments of the method, the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 76 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 77. In particular such embodiments of the method,the nucleic acid sequence encoding the shRNAmiR comprises a sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or more,sequence identity to SEQ ID NO: 86. In further such embodiments of themethod, the nucleic acid sequence encoding the shRNAmiR comprises thesequence of SEQ ID NO: 86.

In some embodiments of the method, the target protein is GR. In somesuch embodiments of the method, cell surface expression of GR is reducedby at least about 10%, about 20%, about 30%, about 40%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or up to about 99% compared to a control cell. Infurther such embodiments, the immune cell is less susceptible to effectsof glucocorticoids (e.g., dexamethasone), such as reduced proliferation.

In some such embodiments of the method, the shRNAmiR has a structurewherein: (a) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 91 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 92; (b) the passenger strand comprises a nucleic acidsequence of SEQ ID NO: 93 and the guide strand comprises a nucleic acidsequence of SEQ ID NO: 94; (c) the passenger strand comprises a nucleicacid sequence of SEQ ID NO: 95 and the guide strand comprises a nucleicacid sequence of SEQ ID NO: 96; (d) the passenger strand comprises anucleic acid sequence of SEQ ID NO: 97 and the guide strand comprises anucleic acid sequence of SEQ ID NO: 98; (e) the passenger strandcomprises a nucleic acid sequence of SEQ ID NO: 99 and the guide strandcomprises a nucleic acid sequence of SEQ ID NO: 100; (f) the passengerstrand comprises a nucleic acid sequence of SEQ ID NO: 101 and the guidestrand comprises a nucleic acid sequence of SEQ ID NO: 102; (g) thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 103 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 104;(h) the passenger strand comprises a nucleic acid sequence of SEQ ID NO:105 and the guide strand comprises a nucleic acid sequence of SEQ ID NO:106; or (i) the passenger strand comprises a nucleic acid sequence ofSEQ ID NO: 107 and the guide strand comprises a nucleic acid sequence ofSEQ ID NO: 108. In particular such embodiments of the method, thepassenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 andthe guide strand comprises a nucleic acid sequence of SEQ ID NO: 96. Inparticular such embodiments of the method, the nucleic acid sequenceencoding the shRNAmiR comprises a sequence having at least 80%, at least85%, at least 90%, at least 95%, or more, sequence identity to SEQ IDNO: 111. In further such embodiments of the method, the nucleic acidsequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 111.

In another aspect, the invention provides an immune cell made by any ofthe methods described herein. In some embodiments, the target protein isbeta-2 microglobulin and the immune cell made by the method has reducedcell-surface expression of beta-2 microglobulin and/or MHC class Iproteins. In some embodiments, the target protein is CS1 and the immunecell made by the method has reduced cell-surface expression of CS1. Insome embodiments, the target protein is TGFRB2, and the immune cell madeby the method has reduced expression of TGFBR2. In some embodiments, thetarget protein is CBL-B, and the immune cell made by the method hasreduced expression of CBL-B. In some embodiments, the target protein isCD52, and the immune cell made by the method has reduced cell-surfaceexpression of CD52. In some embodiments, the target protein is DCK, andthe immune cell made by the method has reduced expression of DCK. Insome embodiments, the target protein is GR, and the immune cell made bythe method has reduced expression of GR.

In another aspect, the invention provides a population of cellscomprising a plurality of the genetically-modified immune cells, or aplurality of the immune cells, described herein. In some embodiments, atleast about 20%, about 30%, about 40%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or up to 100% of cells in the population are thegenetically-modified immune cells described herein, or the immune cellsdescribed herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a plurality ofgenetically-modified immune cells described herein, or a plurality ofthe immune cells described herein. In some embodiments, thepharmaceutical composition comprises a population of cells describedherein.

In another aspect, the invention provides a method of immunotherapy fortreating a disease in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition described herein. In some embodiments, the method is animmunotherapy for the treatment of a cancer in a subject in needthereof, wherein the genetically-modified immune cell, or immune cell,is a genetically-modified human T cell, or a cell derived therefrom, ora genetically-modified NK cell, or a cell derived therefrom, and whereinthe genetically-modified immune cell, or immune cell, comprises a CAR orexogenous TCR, wherein the CAR or the exogenous TCR comprises anextracellular ligand-binding domain having specificity for atumor-specific antigen. In some embodiments of the method, thegenetically-modified immune cell or the immune cell comprises aninactivated TCR alpha gene or an inactivated TCR alpha constant regiongene. In further embodiments of the method, the genetically-modifiedimmune cell, or the immune cell, has no detectable cell-surfaceexpression of an endogenous TCR (e.g., an alpha/beta TCR). In someembodiments of the method, the cancer is selected from the groupconsisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, andleukemia. In certain embodiments of the method, the cancer is selectedfrom the group consisting of a cancer of B cell origin, breast cancer,gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. In particularembodiments of the method, the cancer of B cell origin is selected fromthe group consisting of B-lineage acute lymphoblastic leukemia, B cellchronic lymphocytic leukemia, B cell non-Hodgkin lymphoma, and multiplemyeloma.

In particular embodiments of the method, the subject can be a mammal,such as a human.

In another aspect, the invention provides a method for treating adisease, such as cancer, in a subject in need thereof, wherein themethod comprises administering to the subject a therapeuticallyeffective amount of a population of any genetically-modified immunecells described herein (e.g., a genetically-modified human T cell or NKcell expressing a CAR or exogenous TCR) that comprise in their genome anucleic acid sequence encoding a shRNAmiR that reduces the expression ofendogenous deoxycytidine kinase (DCK), wherein the population ofgenetically-modified immune cells is administered to the subject before,during, or after administration of a purine nucleoside. Reduction of DCKexpression by the shRNAmiR reduces the effect of the purine nucleosideon proliferation or in vivo persistence of the genetically-modifiedimmune cells.

In particular embodiments of the method, the population ofgenetically-modified immune cells and the purine nucleoside areadministered such that the genetically-modified immune cells are presentin the subject (i.e., have not been eliminated by the host) when thepurine nucleoside is administered, or while the purine nucleoside ispresent in the subject at an effective level. In some embodiments of themethod, the purine nucleoside is fludarabine. In some such embodimentsof the method, fludarabine is administered alone or in combination withanother chemotherapeutic compound as part of a lymphodepletion regimenfor immunotherapy.

In particular embodiments of the method, the genetically-modified immunecells are genetically-modified human T cells or genetically-modified NKcells expressing a CAR or exogenous TCR having specificity for anantigen on the targeted cancer cells.

In another aspect, the invention provides a method for treating adisease, such as cancer, in a subject in need thereof, wherein themethod comprises administering to the subject a therapeuticallyeffective amount of a population of any genetically-modified immunecells described herein (e.g., a genetically-modified human T cell or NKcell expressing a CAR or exogenous TCR) that comprise in their genome anucleic acid sequence encoding a shRNAmiR that reduces the expression ofendogenous glucocorticoid receptor (GR), wherein the population ofgenetically-modified immune cells is administered to the subject before,during, or after administration of a corticosteroid. Reduction of GRexpression by the shRNAmiR reduces the effect of the corticosteroid onproliferation or in vivo persistence of the genetically-modified immunecells.

In particular embodiments of the method, the population ofgenetically-modified immune cells and the corticosteroid areadministered such that the genetically-modified immune cells are presentin the subject (i.e., have not been eliminated by the host) when thecorticosteroid is administered, or while the corticosteroid is presentin the subject at an effective level. In some embodiments of the method,the corticosteroid is dexamethasone or methylprednisolone. In some suchembodiments of the method, the corticosteroid is administered alone orin combination with another compound as part of a treatment for reducingcytokine release syndrome during immunotherapy.

In particular embodiments of the method, the genetically-modified immunecells are genetically-modified human T cells or genetically-modified NKcells expressing a CAR or exogenous TCR having specificity for anantigen on the targeted cancer cells.

In another aspect, the invention provides a genetically-modified immunecell or a population thereof, as described herein, or an immune cell ora population thereof, as described herein, for use as a medicament. Theinvention further provides the use of a genetically-modified immune cellor a population thereof, as described herein, or an immune cell or apopulation thereof, as described herein, in the manufacture of amedicament for treating a disease in a subject in need thereof. In onesuch aspect, the medicament is useful in the treatment of a cancer.

In another aspect, the invention provides a genetically-modified cell orpopulation thereof, as described herein, or an immune cell or apopulation thereof, as described herein, for use in treatment of adisease, and preferably in the treatment of a cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows beta-2 microglobulin expression or HLA-A, B, and Cexpression (i.e., MHC class I molecule expression) on T cells transducedwith an AAV comprising construct 7056 which expresses a single copy ofthe shRNA472 in a 3′ to 5′ head-to-tail configuration with the CAR. FIG.1A shows the B2M surface levels in CD3−/CAR+ cells compared tomeganuclease-edited cells expressing no shRNA from a control culture.FIG. 1B shows B2M levels on CD3−/CAR+ versus CD3+/CAR− populations inthe same culture. FIG. 1C shows HLA-ABC (i.e., MHC class I molecule)surface levels in CD3−/CAR+ cells compared to meganuclease-edited cellsexpressing no shRNA from a control culture. FIG. 1D shows HLA-ABC levelson CD3−/CAR+ versus CD3+/CAR− populations in the same culture.

FIG. 2 shows the frequency of CD3−/CAR+ cells, and the knockdown of B2M,in cultures produced with AAV 7056. FIG. 2A shows the frequency ofCD3−/CAR+ cells at day 4. FIG. 2B shows knockdown of B2M at day 4. FIG.2C shows the frequency of CD3−/CAR+ cells at day 7. FIG. 2D showsknockdown of B2M at day 7. FIG. 2E shows the frequency of CD3−/CAR+cells at day 10. FIG. 2F shows knockdown of B2M at day 10.

FIG. 3 shows the frequency of CD3−/CAR+ cells, and the knockdown of B2M,in cultures produced with AAV 7206, AAV 7056, or AAV 7282 three dayspost-transduction. FIG. 3A shows the frequency of CD3−/CAR+ cells in7206-tranduced cells. FIG. 3B shows knockdown of B2M in 7206-tranducedcells. FIG. 3C shows the frequency of CD3−/CAR+ cells in 7282-tranducedcells. FIG. 3D shows knockdown of B2M in 7282-tranduced cells. FIG. 3Eshows the frequency of CD3−/CAR+ cells in 7056-tranduced cells. FIG. 3Fshows knockdown of B2M in 7056-tranduced cells.

FIG. 4 shows the frequency of CD3−/CAR+ cells, and the knockdown of B2M,in cultures produced with AAV 7206, AAV 7056, or AAV 7282 seven dayspost-transduction. FIG. 4A shows the frequency of CD3−/CAR+ cells in7206-tranduced cells. FIG. 4B shows knockdown of B2M in 7206-tranducedcells. FIG. 4C shows the frequency of CD3−/CAR+ cells in 7282-tranducedcells. FIG. 4D shows knockdown of B2M in 7282-tranduced cells. FIG. 4Eshows the frequency of CD3−/CAR+ cells in 7056-tranduced cells. FIG. 4Fshows knockdown of B2M in 7056-tranduced cells.

FIG. 5 shows the frequency of CD3−/CAR+ cells, and the knockdown of B2M,in cultures produced with AAV 7206, AAV 7056, or AAV 7282 eleven dayspost-transduction. FIG. 5A shows the frequency of CD3−/CAR+ cells in7206-tranduced cells. FIG. 5B shows knockdown of B2M in 7206-tranducedcells. FIG. 5C shows the frequency of CD3−/CAR+ cells in 7282-tranducedcells. FIG. 5D shows knockdown of B2M in 7282-tranduced cells. FIG. 5Eshows the frequency of CD3−/CAR+ cells in 7056-tranduced cells. FIG. 5Fshows knockdown of B2M in 7056-tranduced cells.

FIG. 6 shows the effects of B2M knockout or knockdown on the sensitivityof CAR T cells to cytolysis by alloantigen-specific cytotoxiclymphocytes (CTLs) or NK cells. FIG. 6A shows the cytolytic activity ofprimed alloantigen-specific CTLs against B2M knockout and B2M knockdownCAR T cell populations. FIG. 6B shows the cytolytic activity of NK cellsagainst B2M knockout and B2M knockdown CAR T cell populations.

FIG. 7 shows knockout of B2M using an engineered meganuclease andtargeted insertion of a donor template comprising a coding sequence foran HLA-E polypeptide. FIG. 7A shows B2M knockout without targetedinsertion. FIG. 7B shows B2M knockout and targeted insertion of thedonor template using AAV7346.

FIG. 8 shows purification of T cell populations with B2M knockout andcell surface expression of HLA-E encoded by the inserted donor template.FIG. 8A shows the cell population that is B2M-negative. FIG. 8B showsthe purified B2M-negative population. FIG. 8C shows the cell populationthat is HLA-E-positive. FIG. 8D shows the purified HLA-E-positive cellpopulation.

FIG. 9 shows CAR T cell killing by alloantigen-primed CTLs and cellkilling by natural killer (NK) cells. FIG. 9A shows killing ofB2M-positive, B2M-knockout, and B2M-knockout/HLA-E knock in, CAR T cellsby alloantigen-primed CTLs at increasing Effector:Target ratios. FIG. 9Bshows NK cell killing of B2M-positive, B2M-knockout, andB2M-knockout/HLA-E knock in, CAR T cells at increasing Effector:Targetratios.

FIG. 10 shows in vivo efficacy and stability of shRNAmiR-inducedknockdown of B2M in CAR T cells. FIG. 10A shows bioluminescence imagingof total flux over time in mice engrafted with NALM-6 cells and treatedwith vehicle (shown as black line with triangles), mice treated withCD19-directed CAR T cells (shown as dark gray line with circles), ormice treated with CD19-directed CAR T cells with an integratedB2M-targeting shRNAmiR (shown as light gray line with triangles). FIG.10B shows flow cytometry staining for B2M expression on human CD45+cells 14 days after administration of either CD19-directed CAR T cellswith an integrated B2M-targeting shRNAmiR (shown as light grayhistogram) or control CD19-directed CAR T cells (shown as dark grayhistogram).

FIG. 11 shows shRNAmiR-induced stable knockdown of CS1 in CART cells.Three candidate guide and passenger strand sequences for a CS1/SLAMF7shRNAmiR were built into a miR-E scaffold and positioned after the stopcodon of a BCMA-specific CAR. Constructs were designated AAV72101-72103and were used for transduction of donor T cells. FIG. 11A shows CAR andCS1 staining seven days after transduction with an AAV encoding a BCMACAR but no shRNAmiR construct. FIG. 11B shows CAR and CS1 staining sevendays after transduction with AAV 72101. FIG. 11C shows CAR and CS1staining seven days after transduction with AAV 72102. FIG. 11D showsCAR and CS1 staining seven days after transduction with AAV 72103.

FIG. 12 shows shRNAmiR-induced stable knockdown of TGFRB2 in CART cells.Multiple candidate guide and passenger strand sequences for a TGFBR2shRNAmiR were built into a miR-E scaffold and positioned after the stopcodon of a CD19-specific CAR. Constructs were designated AAV 72110-72114and were used for transduction of donor T cells. FIG. 12A shows CAR andTGFBR2 staining after mock transduction. FIG. 12B shows CAR and TGFBR2staining at day 14 post-transduction with AAV 72110. FIG. 12C shows CARand TGFBR2 staining at day 14 post-transduction with AAV 72111. FIG. 12Dshows CAR and TGFBR2 staining at day 14 post-transduction with AAV72112. FIG. 12E shows CAR and TGFBR2 staining at day 14post-transduction with AAV 72113. FIG. 12F shows CAR and TGFBR2 stainingat day 14 post-transduction with AAV 72114.

FIG. 13 shows flow cytometry detecting knockout of TGFBR2 in T cellsusing two engineered meganucleases. FIG. 13A shows a negative stainingcontrol. FIG. 13B shows mock-transfected T cells. FIG. 13C shows T cellstransfected with mRNA encoding the TGF 1-2x.5 meganuclease. FIG. 13Dshows T cells transfected with mRNA encoding the TGF 1-2L.296meganuclease.

FIG. 14 shows flow cytometry staining for phosphorylated SMAD 2/3 inTGFBR2-positive and negative T cells treated with TGFB1.

FIG. 15 shows flow cytometry for phosphorylated SMAD 2/3 inTGFBR2-positive CAR T cells, CAR T cells expressing an anti-TGFBR2shRNAmiR to knockdown protein expression, and T cells treated with anengineered meganuclease to knockout TGFBR2 expression.

FIG. 16 shows flow cytometry for phosphorylated SMAD 2/3 in untreatedcontrol BCMA CAR T cells (A), BCMA CAR T cells treated with TGFβ1 (B),BCMA CAR T cells treated with TGFβ1 having either TGFBR2 knocked outwith the TGF 1-2L.296 meganuclease (C) or TGFBR2 knocked down with the72112 TGFBR2 shRNAmiR (D).

FIG. 17 shows BCMA CAR T cell numbers over time following co-culturewith normal K562 cells, K562 cells transfected to stably express BCMA(KBCMA), or K562 cells stably expressing BCMA and constitutivelysecreting active TGFβ31 (KBCMA-TGF). BCMA CAR T cells were modified toknock down TGFBR2 using a shRNAmiR (TGFBRKD) or modified to knock downB2M with a shRNAmiR (CtrlKD).

FIG. 18 shows target cell numbers over time following co-culture of BCMACAR T cells with K562 cells transfected to stably express BCMA (KBCMA),or K562 cells stably expressing BCMA and constitutively secreting activeTGFβ31 (KBCMA-TGF). BCMA CAR T cells were modified to knock down TGFBR2using a shRNAmiR (TGFBRKD) or modified to knock down B2M with a shRNAmiR(CtrlKD).

FIG. 19 shows BCMA CAR T cell numbers over time following co-culturewith normal K562 cells, K562 cells transfected to stably express BCMA,and K562 cells stably expressing BCMA and constitutively secretingactive TGFβ1. BCMA CAR T cells were modified to knock down TGFBR2 usinga shRNAmiR (72154), modified to knock down B2M with a shRNAmiR (72155),or modified to knockout TGFBR2 with an engineered meganuclease (dKO).

FIG. 20 shows the CD4:CD8 ratio of BCMA CAR T cells over time followingco-culture with K562 cells transfected to stably express BCMA, and K562cells stably expressing BCMA and constitutively secreting active TGFβ1.BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR(72154), modified to knock down B2M with a shRNAmiR (72155), or modifiedto knockout TGFBR2 with an engineered meganuclease (dKO).

FIG. 21 shows BCMA CAR T cell numbers over time following co-culturewith normal U266 cells, or U266 cells constitutively secreting activeTGFβ1. BCMA CAR T cells were modified to knock down TGFBR2 using ashRNAmiR (72154), modified to knock down B2M with a shRNAmiR (72155), ormodified to knockout TGFBR2 with an engineered meganuclease (dKO).

FIG. 22 shows BCMA CAR T cell numbers over time following co-culturewith U266 cells, or U266 cells constitutively secreting active TGFβ1.BCMA CART cells were modified to knock down TGFBR2 using a shRNAmiR(72154), modified to knock down B2M with a shRNAmiR (72155), or modifiedto knockout TGFBR2 with an engineered meganuclease (dKO).

FIG. 23 shows flow cytometry plots of the number of CD4+ CAR T cells atdifferent time points in co-culture with U266 cells. FIG. 23A shows CART cells incorporating the 72154 construct. FIG. 23B shows CAR T cellsincorporating the 72155 construct. FIG. 23C shows CAR T cells having anHLA-E fusion protein knocked into the B2M gene.

FIG. 24 shows flow cytometry plots of live versus dead U266 cells afterco-culture with BCMA CAR T cell variants for 16 days. FIG. 24A showsco-culture of U266 cells with BCMA CAR T cells modified to knock downTGFBR2 using a shRNAmiR (TGFbRKD). FIG. 24B shows co-culture of U266cells with BCMA CAR T cells modified to knock down B2M using a shRNAmiR(Ctrl KD). FIG. 24C shows co-culture of U266 cells with BCMA CAR T cellsmodified to knockout TGFBR2 with an engineered meganuclease (TGFbR KO).FIG. 24D shows co-culture of U266 cells secreting active TGFβ1 with BCMACAR T cells modified to knock down TGFBR2 using a shRNAmiR (TGFbRKD).FIG. 24E shows co-culture of U266 cells secreting active TGFβ1 with BCMACAR T cells modified to knock down B2M using a shRNAmiR (Ctrl KD). FIG.24F shows co-culture of U266 cells secreting active TGFβ1 with BCMA CART cells modified to knockout TGFBR2 with an engineered meganuclease(TGFbR KO).

FIG. 25 shows shRNAmiR-induced stable knockdown of CD52 in CAR T cells.Multiple candidate guide and passenger strand sequences for a CD52shRNAmiR were built into a miR-E scaffold and positioned after the stopcodon of a CD19-specific CAR. Constructs were designated AAV 72123 andAAV 72124 and were used for transduction of donor T cells. FIG. 25Ashows staining of CD3−/CAR+ T cell populations following AAVtransduction. FIG. 25B shows knockdown of CD52 in CD3−/CAR+ T cells 10days after transduction with AAV 72123. FIG. 25C shows knockdown of CD52in CD3−/CAR+ T cells 10 days after transduction with AAV 72124.

FIG. 26 shows flow cytometry staining for B2M and CD52 in T cellsexpressing a B2M-targeting shRNAmiR and a CD52-targeting shRNAmiR.

FIG. 27 shows the percentage of CAR+ T cells recovered following CD52depletion of CAR T populations expressing a B2M-targeting shRNAmiR and aCD52-targeting shRNAmiR.

FIG. 28 shows diagrams of constructs 73161, 73162, 73163, and 73164.FIG. 28A shows construct 73161 which comprises a JeT promoter, a CD19CAR gene, P2 A/furin site, an HLA-E gene comprising a synthetic intronwhich comprises a B2M-targeting shRNAmiR, and an SV40 bi-directionalpolyA sequence. FIG. 28B shows construct 73162 which comprises a JeTpromoter, a CD19 CAR gene comprising a synthetic intron which comprisesa B2M-targeting shRNAmiR, an SV40 bi-directional polyA sequence, asecond JeT promoter, an HLA-E gene, and a bovine growth hormone (BGH)termination signal. FIG. 28C shows construct 73163 which comprises a JeTpromoter, a CD19 CAR gene, an SV40 bi-directional polyA sequence, an EF1alpha core promoter, an HLA-E gene comprising a synthetic intron whichcomprises a B2M-targeting shRNAmiR, and a BGH termination signal. FIG.28D shows the 73164 construct which comprises a JeT promoter, a CD19 CARgene, an SV40 bi-directional polyA sequence, a second JeT promoter, anHLA-E gene comprising a synthetic intron which comprises a B2M-targetingshRNAmiR, and a BGH termination signal.

FIG. 29 shows a table summarizing the CAR phenotype of T cells in whichthe identified constructs were introduced by AAV and inserted into theTRAC locus only (7206 and 73161-73164), or cells in which a CAR gene wasinserted in the TRAC locus and an HLA-E gene was inserted in the B2Mlocus (dKO dKI). The table provides the percentage of cells that wereCD3−/CAR+, percentage of CD3 knockout cells that had a CAR knock-in,mean fluorescence intensity (MFI) of the expressed CAR, and comparisonof the MFI of each CAR when compared to the CAR introduced using the7206 construct.

FIG. 30 shows a table summarizing the HLA-ABC and HLA-E phenotypes of Tcells in which the identified constructs were introduced by AAV andinserted into the TRAC locus only (7206 and 73161-73164), or cells inwhich a CAR gene was inserted in the TRAC locus and an HLA-E gene wasinserted in the B2M locus (dKO dKI). The table provides the percentageof HLA-ABC expression compared to wild-type in the CD3−/CAR+ population,the percentage of HLA-ABC knockdown with the wild-type gated out, thepercentage of cells expressing HLA-E in the CD3−/CAR+ population, theMFI of HLA-E expression in such cells, and the percentage of cells inthe CAR− population that were HLA-E+.

FIG. 31 shows a table summarizing characteristics of CAR T cells inwhich the identified constructs were introduced by AAV and inserted intothe TRAC locus only (7206 and 73161-73164), or cells in which a CAR genewas inserted in the TRAC locus and an HLA-E gene was inserted in the B2Mlocus (dKO dKI).

FIG. 32 shows cytolysis of CD19 CAR T variants prepared with T cellsfrom a first donor (HC6366) when co-cultured with alloantigen-primedCTLs from two different donors (K3212 or K2916). FIG. 32A shows killingof CART cells by K3212 alloantigen-primed CTLs. FIG. 32B shows killingof CART cells by K2916 alloantigen-primed CTLs.

FIG. 33 shows natural killer (NK) cell cytolysis of CD19 CART variantsin co-culture at multiple time points at a 1:1 ratio. FIG. 33A showscytolysis at 24 hours. FIG. 33B shows cytolysis at 48 hours. FIG. 33Cshows cytolysis at 120 hours.

FIG. 34 shows luminescence measurements demonstrating in vivo efficacyof CD19 CAR T variants in immunodeficient mice engrafted with NALM/6leukemia cells.

FIG. 35 shows survival of immunodeficient mice engrafted with NALM/6leukemia cells treated with CD19 CAR T variants.

FIG. 36 shows changes in the percent knock in of a CD19 CAR sequence,with or without a DCK shRNAmiR, in CD3 knockout T cells followingincubation with different concentrations of fludarabine.

FIG. 37 shows events/uL of CD3−/CAR+ population versus the concentrationof fludarabine incubated with CD19 CAR T cell variants that include, ordo not include, a DCK shRNAmiR.

FIG. 38 shows the number of viable cells/mL on day 4 followingpost-treatment of different CD19 CAR T cell variants that include, or donot include, a DCK shRNAmiR, with different concentrations offludarabine.

FIG. 39 shows the number of viable cells/mL on day 8 followingpost-treatment of different CD19 CAR T cell variants that include, or donot include, a DCK shRNAmiR, with different concentrations offludarabine.

FIG. 40 shows the percent cytolysis of CD19-expressing HEK293 cells(targets) co-cultured with CD19 CAR T cells (effectors) lacking a DCKshRNAmiR in the presence of different concentrations of fludarabine.Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4.

FIG. 41 shows the percent cytolysis of CD19-expressing HEK293 cells(targets) co-cultured with CD19 CAR T cells (effectors) comprising a DCKshRNAmiR (72138) in the presence of different concentrations offludarabine. Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4.

FIG. 42 shows the percent cytolysis of CD19-expressing HEK293 cells(targets) co-cultured with CD19 CAR T cells (effectors) comprising a DCKshRNAmiR (72136) in the presence of different concentrations offludarabine. Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4.

FIG. 43 shows changes in the percent knock in of a CD19 CAR sequence,with or without a GR shRNAmiR, in CD3 knockout T cells followingincubation with different concentrations of dexamethasone.

FIG. 44 shows events/uL of CD3−/CAR+ population versus the concentrationof dexamethasone incubated with CD19 CAR T cell variants that include,or do not include, a GR shRNAmiR.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the nucleic acid sequence of the 5′ miR-Escaffold domain coding sequence.

SEQ ID NO: 2 sets forth the nucleic acid sequence of the 5′ mir-E basalstem domain coding sequence.

SEQ ID NO: 3 sets forth the nucleic acid sequence of the miR-30a loopdomain coding sequence.

SEQ ID NO: 4 sets forth the nucleic acid sequence of the 3′ miR-E basalstem domain coding sequence.

SEQ ID NO: 5 sets forth the nucleic acid sequence of the 3′ miR-Escaffold domain coding sequence.

SEQ ID NO: 6 sets forth the nucleic acid sequence encoding the shRNA472.

SEQ ID NO: 7 sets forth the nucleic acid sequence encoding the passengerstrand of the 7282 beta-2 microglobulin (B2M) shRNAmiR.

SEQ ID NO: 8 sets forth the nucleic acid sequence encoding the guidestrand of the 7282 B2M shRNAmiR.

SEQ ID NO: 9 sets forth the nucleic acid sequence encoding the passengerstrand of the 7285 B2M shRNAmiR.

SEQ ID NO: 10 sets forth the nucleic acid sequence encoding the guidestrand of the 7285 B2M shRNAmiR.

SEQ ID NO: 11 sets forth the nucleic acid sequence encoding thepassenger strand of the 7286 B2M shRNAmiR.

SEQ ID NO: 12 sets forth the nucleic acid sequence encoding the guidestrand of the 7286 B2M shRNAmiR.

SEQ ID NO: 13 sets forth the nucleic acid sequence encoding thepassenger strand of the 7287 B2M shRNAmiR.

SEQ ID NO: 14 sets forth the nucleic acid sequence encoding the guidestrand of the 7287 B2M shRNAmiR.

SEQ ID NO: 15 sets forth the nucleic acid sequence encoding thepassenger strand of the 7288 B2M shRNAmiR.

SEQ ID NO: 16 sets forth the nucleic acid sequence encoding the guidestrand of the 7288 B2M shRNAmiR.

SEQ ID NO: 17 sets forth the nucleic acid sequence encoding thepassenger strand of the 7289 B2M shRNAmiR.

SEQ ID NO: 18 sets forth the nucleic acid sequence encoding the guidestrand of the 7289 B2M shRNAmiR.

SEQ ID NO: 19 sets forth the nucleic acid sequence encoding thepassenger strand of the 7290 B2M shRNAmiR.

SEQ ID NO: 20 sets forth the nucleic acid sequence encoding the guidestrand of the 7290 B2M shRNAmiR.

SEQ ID NO: 21 sets forth the nucleic acid sequence encoding thepassenger strand of the 72101 CS1 shRNAmiR.

SEQ ID NO: 22 sets forth the nucleic acid sequence encoding the guidestrand of the 72101 CS1 shRNAmiR.

SEQ ID NO: 23 sets for the nucleic acid sequence encoding the passengerstrand of the 72102 CS1 shRNAmiR.

SEQ ID NO: 24 sets for the nucleic acid sequence encoding the guidestrand of the 72102 CS1 shRNAmiR.

SEQ ID NO: 25 sets for the nucleic acid sequence encoding the passengerstrand of the 72103 CS1 shRNAmiR.

SEQ ID NO: 26 sets forth the nucleic acid sequence encoding the guidestrand of the 72103 CS1 shRNAmiR.

SEQ ID NO: 27 sets forth the nucleic acid sequence encoding thepassenger strand of the 72110 transforming growth factor beta receptor 2(TGFBR2) shRNAmiR.

SEQ ID NO: 28 sets forth the nucleic acid sequence encoding the guidestrand of the 72110 TGFBR2 shRNAmiR.

SEQ ID NO: 29 sets forth the nucleic acid sequence encoding thepassenger strand of the 72111 TGFBR2 shRNAmiR.

SEQ ID NO: 30 sets forth the nucleic acid sequence encoding the guidestrand of the 72111 TGFBR2 shRNAmiR.

SEQ ID NO: 31 sets forth the nucleic acid sequence encoding thepassenger strand of the 72112 TGFBR2 shRNAmiR.

SEQ ID NO: 32 sets forth the nucleic acid sequence encoding the guidestrand of the 72112 TGFBR2 shRNAmiR.

SEQ ID NO: 33 sets forth the nucleic acid sequence encoding thepassenger strand of the 72113 TGFBR2 shRNAmiR.

SEQ ID NO: 34 sets forth the nucleic acid sequence encoding the guidestrand of the 72113 TGFBR2 shRNAmiR.

SEQ ID NO: 35 sets forth the nucleic acid sequence encoding thepassenger strand of the 72114 TGFBR2 shRNAmiR.

SEQ ID NO: 36 sets forth the nucleic acid sequence encoding the guidestrand of the 72114 TGFBR2 shRNAmiR.

SEQ ID NO: 37 sets forth the nucleic acid sequence encoding thepassenger strand of the 72123 CD52 shRNAmiR.

SEQ ID NO: 38 sets forth the nucleic acid sequence encoding the guidestrand of the 72123 CD52 shRNAmiR.

SEQ ID NO: 39 sets forth the nucleic acid sequence encoding thepassenger strand of the 72124 CD52 shRNAmiR.

SEQ ID NO: 40 sets forth the nucleic acid sequence encoding the guidestrand of the 72124 CD52 shRNAmiR.

SEQ ID NO: 41 sets forth the nucleic acid sequence encoding the 7282 B2MshRNAmiR.

SEQ ID NO: 42 sets forth the nucleic acid sequence encoding the 7285 B2MshRNAmiR.

SEQ ID NO: 43 sets forth the nucleic acid sequence encoding the 7286 B2MshRNAmiR.

SEQ ID NO: 44 sets forth the nucleic acid sequence encoding the 7287 B2MshRNAmiR.

SEQ ID NO: 45 sets forth the nucleic acid sequence encoding the 7288 B2MshRNAmiR.

SEQ ID NO: 46 sets forth the nucleic acid sequence encoding the 7289 B2MshRNAmiR.

SEQ ID NO: 47 sets forth the nucleic acid sequence encoding the 7290 B2MshRNAmiR.

SEQ ID NO: 48 sets forth the nucleic acid sequence encoding the 72101CS1 shRNAmiR.

SEQ ID NO: 49 sets forth the nucleic acid sequence encoding the 72102CS1 shRNAmiR.

SEQ ID NO: 50 sets forth the nucleic acid sequence encoding the 72103CS1 shRNAmiR.

SEQ ID NO: 51 sets forth the nucleic acid sequence encoding the 72110TGFBR2 shRNAmiR.

SEQ ID NO: 52 sets forth the nucleic acid sequence encoding the 72111TGFBR2 shRNAmiR.

SEQ ID NO: 53 sets forth the nucleic acid sequence encoding the 72112TGFBR2 shRNAmiR.

SEQ ID NO: 54 sets forth the nucleic acid sequence encoding the 72113TGFBR2 shRNAmiR.

SEQ ID NO: 55 sets forth the nucleic acid sequence encoding the 72114TGFBR2 shRNAmiR.

SEQ ID NO: 56 sets forth the nucleic acid sequence encoding the 72123CD52 shRNAmiR.

SEQ ID NO: 57 sets forth the nucleic acid sequence encoding the 72124CD52 shRNAmiR.

SEQ ID NO: 58 sets forth the nucleic acid sequence of the TRC 1-2recognition sequence (sense).

SEQ ID NO: 59 sets forth the nucleic acid sequence of the TRC 1-2recognition sequence (antisense).

SEQ ID NO: 60 sets forth the nucleic acid sequence of the B2M 13-14recognition sequence (sense).

SEQ ID NO: 61 sets forth the nucleic acid sequence of the B2M 13-14recognition sequence (antisense).

SEQ ID NO: 62 sets forth the nucleic acid sequence of the TGF 1-2recognition sequence (sense).

SEQ ID NO: 63 sets forth the nucleic acid sequence of the TGF 1-2recognition sequence (antisense).

SEQ ID NO: 64 sets forth the amino acid sequence of the TGF 1-2x.5meganuclease.

SEQ ID NO: 65 sets forth the amino acid sequence of the TGF 1-2L.296meganuclease.

SEQ ID NO: 66 sets forth the amino acid sequence of an HLA class Ihistocompatibility antigen, alpha chain E (HLA-E) fusion polypeptide.

SEQ ID NO: 67 sets forth the nucleic acid sequence of a JeT promoter.

SEQ ID NO: 68 sets forth the nucleic acid sequence of a bidirectionalSV40 polyA signal.

SEQ ID NO: 69 sets forth the nucleic acid sequence of a syntheticintron.

SEQ ID NO: 70 sets forth the nucleic acid sequence of a P2 A/furin site.

SEQ ID NO: 71 sets forth the nucleic acid sequence of a bovine growthhormone termination signal.

SEQ ID NO: 72 sets forth the nucleic acid sequence of an EF1 alpha corepromoter.

SEQ ID NO: 73 sets forth the amino acid sequence of a signal peptide.

SEQ ID NO: 74 sets forth the nucleic acid sequence of a cassettecomprised by construct 73161.

SEQ ID NO: 75 sets forth the nucleic acid sequence of a cassettecomprised by construct 73163.

SEQ ID NO: 76 sets forth the nucleic acid sequence encoding thepassenger strand of the 72136 DCK shRNAmiR.

SEQ ID NO: 77 sets forth the nucleic acid sequence encoding the guidestrand of the 72136 DCK shRNAmiR.

SEQ ID NO: 78 sets forth the nucleic acid sequence encoding thepassenger strand of the 72137 DCK shRNAmiR.

SEQ ID NO: 79 sets forth the nucleic acid sequence encoding the guidestrand of the 72137 DCK shRNAmiR.

SEQ ID NO: 80 sets forth the nucleic acid sequence encoding thepassenger strand of the 72138 DCK shRNAmiR.

SEQ ID NO: 81 sets forth the nucleic acid sequence encoding the guidestrand of the 72138 DCK shRNAmiR.

SEQ ID NO: 82 sets forth the nucleic acid sequence encoding thepassenger strand of the 72139 DCK shRNAmiR.

SEQ ID NO: 83 sets forth the nucleic acid sequence encoding the guidestrand of the 72139 DCK shRNAmiR.

SEQ ID NO: 84 sets forth the nucleic acid sequence encoding thepassenger strand of the 72140 DCK shRNAmiR.

SEQ ID NO: 85 sets forth the nucleic acid sequence encoding the guidestrand of the 72140 DCK shRNAmiR.

SEQ ID NO: 86 sets forth the nucleic acid sequence encoding the 72136DCK shRNAmiR.

SEQ ID NO: 87 sets forth the nucleic acid sequence encoding the 72137DCK shRNAmiR.

SEQ ID NO: 88 sets forth the nucleic acid sequence encoding the 72138DCK shRNAmiR.

SEQ ID NO: 89 sets forth the nucleic acid sequence encoding the 72139DCK shRNAmiR.

SEQ ID NO: 90 sets forth the nucleic acid sequence encoding the 72140DCK shRNAmiR.

SEQ ID NO: 91 sets forth the nucleic acid sequence encoding thepassenger strand of the 72142 GR shRNAmiR.

SEQ ID NO: 92 sets forth the nucleic acid sequence encoding the guidestrand of the 72142 GR shRNAmiR.

SEQ ID NO: 93 sets forth the nucleic acid sequence encoding thepassenger strand of the 72143 GR shRNAmiR.

SEQ ID NO: 94 sets forth the nucleic acid sequence encoding the guidestrand of the 72143 GR shRNAmiR.

SEQ ID NO: 95 sets forth the nucleic acid sequence encoding thepassenger strand of the 72145 GR shRNAmiR.

SEQ ID NO: 96 sets forth the nucleic acid sequence encoding the guidestrand of the 72145 GR shRNAmiR.

SEQ ID NO: 97 sets forth the nucleic acid sequence encoding thepassenger strand of the 72146 GR shRNAmiR.

SEQ ID NO: 98 sets forth the nucleic acid sequence encoding the guidestrand of the 72146 GR shRNAmiR.

SEQ ID NO: 99 sets forth the nucleic acid sequence encoding thepassenger strand of the 72148 GR shRNAmiR.

SEQ ID NO: 100 sets forth the nucleic acid sequence encoding the guidestrand of the 72148 GR shRNAmiR.

SEQ ID NO: 101 sets forth the nucleic acid sequence encoding thepassenger strand of the 72149 GR shRNAmiR.

SEQ ID NO: 102 sets forth the nucleic acid sequence encoding the guidestrand of the 72149 GR shRNAmiR.

SEQ ID NO: 103 sets forth the nucleic acid sequence encoding thepassenger strand of the 72150 GR shRNAmiR.

SEQ ID NO: 104 sets forth the nucleic acid sequence encoding the guidestrand of the 72150 GR shRNAmiR.

SEQ ID NO: 105 sets forth the nucleic acid sequence encoding thepassenger strand of the 72151 GR shRNAmiR.

SEQ ID NO: 106 sets forth the nucleic acid sequence encoding the guidestrand of the 72151 GR shRNAmiR.

SEQ ID NO: 107 sets forth the nucleic acid sequence encoding thepassenger strand of the 72152 GR shRNAmiR.

SEQ ID NO: 108 sets forth the nucleic acid sequence encoding the guidestrand of the 72152 GR shRNAmiR.

SEQ ID NO: 109 sets forth the nucleic acid sequence encoding the 72142GR shRNAmiR.

SEQ ID NO: 110 sets forth the nucleic acid sequence encoding the 72143GR shRNAmiR.

SEQ ID NO: 111 sets forth the nucleic acid sequence encoding the 72145GR shRNAmiR.

SEQ ID NO: 112 sets forth the nucleic acid sequence encoding the 72146GR shRNAmiR.

SEQ ID NO: 113 sets forth the nucleic acid sequence encoding the 72148GR shRNAmiR.

SEQ ID NO: 114 sets forth the nucleic acid sequence encoding the 72149GR shRNAmiR.

SEQ ID NO: 115 sets forth the nucleic acid sequence encoding the 72150GR shRNAmiR.

SEQ ID NO: 116 sets forth the nucleic acid sequence encoding the 72151GR shRNAmiR.

SEQ ID NO: 117 sets forth the nucleic acid sequence encoding the 72152GR shRNAmiR.

SEQ ID NO: 118 sets forth the amino acid sequence of an HLA-E-01:03protein.

SEQ ID NO: 119 sets forth the amino acid sequence of a beta-2microglobulin protein.

SEQ ID NO: 120 sets forth the amino acid sequence of an HLA-G leaderpeptide.

SEQ ID NO: 121 sets forth the amino acid sequence of a (GGGGS)3 linkerpeptide.

SEQ ID NO: 122 sets forth the amino acid sequence of a (GGGGS)4 linkerpeptide.

SEQ ID NO: 123 sets forth the amino acid sequence of a wild-type I-CreIhoming endonuclease from Chlamydomonas reinhardtii.

DETAILED DESCRIPTION OF THE INVENTION

1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

As used herein, the terms “exogenous” or “heterologous” in reference toa nucleotide sequence or amino acid sequence are intended to mean asequence that is purely synthetic, that originates from a foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention.

As used herein, the term “endogenous” in reference to a nucleotidesequence or protein is intended to mean a sequence or protein that isnaturally comprised within or expressed by a cell.

As used herein, the terms “nuclease” and “endonuclease” are usedinterchangeably to refer to naturally-occurring or engineered enzymeswhich cleave a phosphodiester bond within a polynucleotide chain.

As used herein, the term “shRNA” or “short hairpin RNA” refers to anartificial RNA molecule comprising a hairpin that can be used to silencegene expression via RNA interference.

As used herein, the term “miRNA” or “microRNA” or “miR” refers to maturemicroRNAs (miRNAs) that are endogenously encoded ˜22 nt long RNAs thatpost-transcriptionally reduce the expression of target genes. miRNAs arefound in plants, animals, and some viruses and are generally expressedin a highly tissue- or developmental-stage-specific fashion.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (stem portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion), In some cases, the loop may also be very short and thereby notbe recognized by Dicer, leading to Dicer-independent shRNAs (comparableto the endogenous miR0431). The term “hairpin” is also used herein torefer to stem-loop structures. The actual primary sequence ofnucleotides within the stem-loop structure is not critical to thepractice of the description as long as the secondary structure ispresent. As is known in the art, the secondary structure does notrequire exact base-pairing. Thus, the stem may include one or more basemismatches. Alternatively, the base-pairing may be exact (i.e., notinclude any mismatches).

As used herein, the terms “shRNAmiR” and “microRNA-adapted shRNA” referto shRNA sequences embedded within a microRNA scaffold. A shRNAmiRmolecule mimics naturally-occurring pri-miRNA molecules in that theycomprise a hairpin flanked by sequences necessary for efficientprocessing and can be processed by the Drosha enzyme into pre-miRNAs,exported into the cytoplasm, and cleaved by Dicer, after which themature miRNA can enter the RISC. The microRNA scaffold can be derivedfrom naturally-occurring microRNA, pre-miRNAs, or pri-miRNAs or variantsthereof. In some embodiments, the shRNA sequences which the shRNAmiR isbased upon is of a different length from miRNAs (which are 22nucleotides long) and the miRNA scaffold must therefore be modified inorder to accommodate the longer or shorter shRNA sequence length.

As used herein, the term “microRNA flanking sequences” refers tonucleotide sequences comprising microRNA processing elements. MicroRNAprocessing elements are the minimal nucleic acid sequences whichcontribute to the production of mature microRNA from primary microRNA orprecursor microRNA. Often these elements are located within a 40nucleotide sequence that flanks a microRNA stem-loop structure. In someinstances, the microRNA processing elements are found within a stretchof nucleotide sequences of between 5 and 4,000 nucleotides in lengththat flank a microRNA stem-loop structure. MicroRNA flanking sequencesused in the shRNAmiR molecules can be naturally-occurring sequencesflanking naturally-occurring microRNA or can be variants thereof.MicroRNA flanking sequences include miR scaffold domains and miR basalstem domains.

shRNAmiR molecules used in the presently disclosed compositions andmethods can comprise in the 5′ to 3′ direction: (a) a 5′ miR scaffolddomain; (b) a 5′ miR basal stem domain; (c) a passenger strand; (d) amiR loop domain; (e) a guide strand; (f) a 3′ miR basal stem domain; and(g) a 3′ miR scaffold domain.

As used herein, the term “miR scaffold domain” as it relates to ashRNAmiR refers to a nucleotide sequence that can flank either the 5′ or3′ end of a microRNA or shRNA in a shRNAmiR molecule and can be derivedfrom a naturally-occurring microRNA flanking sequence or a variantthereof. In general, the miR basal stem domain sequence separates theshRNA sequence (passenger and guide strand, and miR loop domain) and thescaffold domains. The 5′ miR scaffold domain can comprise a restrictionenzyme (e.g., type IIS restriction enzyme) recognition sequence at ornear its 3′ end and the 3′ miR scaffold domain can comprise arestriction enzyme recognition sequence at or near its 5′ end, thusfacilitating the insertion of a shRNA sequence. In some embodiments, thesecondary structure of the miR scaffold domain is more important thanthe actual sequence thereof.

As used herein, the term “miR basal stem domain” as it relates to ashRNAmiR refers to sequences immediately flanking the passenger andguide strand sequences that comprise the base of the hairpin stem belowthe passenger:guide duplex. Thus, the 5′ and 3′ miR basal stem domainsare complementary (fully or partially) in sequence to one another. Insome embodiments, the 5′ and 3′ miR basal stem domains comprisesequences that when hybridized together, form two mismatch bubbles, eachcomprising one or two mismatched base pairs.

As used herein, the term “passenger strand” as it relates to a shRNAmiRrefers to the sequence of the shRNAmiR, which is complementary (fully orpartially) to the guide sequence.

As used herein, the term “guide strand” as it relates to a shRNAmiRrefers to the sequence of the shRNAmiR that has complementarity (full orpartial) with the target mRNA sequence for which a reduction inexpression is desired.

As used herein, a “miR loop domain” as it relates to a shRNAmiR refersto the single-stranded loop sequence at one end of the passenger:guideduplex of the shRNAmiR. The miR loop domain can be derived from anaturally-occurring pre-microRNA loop sequence or a variant thereof.

As used herein, the terms “cleave” or “cleavage” refer to the hydrolysisof phosphodiester bonds within the backbone of a recognition sequencewithin a target sequence that results in a double-stranded break withinthe target sequence, referred to herein as a “cleavage site”.

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. In some embodiments, the recognition sequence for ameganuclease of the present disclosure is 22 base pairs. A meganucleasecan be an endonuclease that is derived from I-CreI (SEQ ID NO: 123), andcan refer to an engineered variant of I-CreI that has been modifiedrelative to natural I-CreI with respect to, for example, DNA-bindingspecificity, DNA cleavage activity, DNA-binding affinity, ordimerization properties. Methods for producing such modified variants ofI-CreI are known in the art (e.g., WO 2007/047859, incorporated byreference in its entirety). A meganuclease as used herein binds todouble-stranded DNA as a heterodimer. A meganuclease may also be a“single-chain meganuclease” in which a pair of DNA-binding domains isjoined into a single polypeptide using a peptide linker. The term“homing endonuclease” is synonymous with the term “meganuclease.”Meganucleases of the present disclosure are substantially non-toxic whenexpressed in cells, particularly in human immune cells, such that cellscan be transfected and maintained at 37° C. without observingdeleterious effects on cell viability or significant reductions inmeganuclease cleavage activity when measured using the methods describedherein.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of nuclease subunits joined by a linker. Asingle-chain meganuclease has the organization: N-terminalsubunit—Linker—C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will bindnon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two nuclease subunits into a single polypeptide. Alinker may have a sequence that is found in natural proteins or may bean artificial sequence that is not found in any natural protein. Alinker may be flexible and lacking in secondary structure or may have apropensity to form a specific three-dimensional structure underphysiological conditions. A linker can include, without limitation,those encompassed by U.S. Pat. Nos. 8,445,251, 9,340,777, 9,434,931, and10,041,053, each of which is incorporated by reference in its entirety.In some embodiments, a linker may have at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, ormore, sequence identity to residues 154-195 of SEQ ID NO: 65. In someembodiments, a linker may have an amino acid sequence comprisingresidues 154-195 of SEQ ID NO: 65.

As used herein, the term “TALEN” refers to an endonuclease comprising aDNA-binding domain comprising a plurality of TAL domain repeats fused toa nuclease domain or an active portion thereof from an endonuclease orexonuclease, including but not limited to a restriction endonuclease,homing endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAseI, micrococcal nuclease, and yeast HO endonuclease. See, for example,Christian et al. (2010) Genetics 186:757-761, which is incorporated byreference in its entirety. Nuclease domains useful for the design ofTALENs include those from a Type IIs restriction endonuclease, includingbut not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI,BglI, and AlwI. Additional Type IIs restriction endonucleases aredescribed in International Publication No. WO 2007/014275, which isincorporated by reference in its entirety. In some embodiments, thenuclease domain of the TALEN is a FokI nuclease domain or an activeportion thereof. TAL domain repeats can be derived from the TALE(transcription activator-like effector) family of proteins used in theinfection process by plant pathogens of the Xanthomonas genus. TALdomain repeats are 33-34 amino acid sequences with divergent 12th and13th amino acids. These two positions, referred to as the repeatvariable dipeptide (RVD), are highly variable and show a strongcorrelation with specific nucleotide recognition. Each base pair in theDNA target sequence is contacted by a single TAL repeat with thespecificity resulting from the RVD. In some embodiments, the TALENcomprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires twoDNA recognition regions (i.e., “half-sites”) flanking a nonspecificcentral region (i.e., the “spacer”). The term “spacer” in reference to aTALEN refers to the nucleic acid sequence that separates the two nucleicacid sequences recognized and bound by each monomer constituting aTALEN. The TAL domain repeats can be native sequences from anaturally-occurring TALE protein or can be redesigned through rationalor experimental means to produce a protein that binds to apre-determined DNA sequence (see, for example, Boch et al. (2009)Science 326(5959):1509-1512 and Moscou and Bogdanove (2009) Science326(5959):1501, each of which is incorporated by reference in itsentirety). See also, U.S. Publication No. 20110145940 and InternationalPublication No. WO 2010/079430 for methods for engineering a TALEN torecognize and bind a specific sequence and examples of RVDs and theircorresponding target nucleotides. In some embodiments, each nuclease(e.g., FokI) monomer can be fused to a TAL effector sequence thatrecognizes and binds a different DNA sequence, and only when the tworecognition sites are in close proximity do the inactive monomers cometogether to create a functional enzyme. It is understood that the term“TALEN” can refer to a single TALEN protein or, alternatively, a pair ofTALEN proteins (i.e., a left TALEN protein and a right TALEN protein)which bind to the upstream and downstream half-sites adjacent to theTALEN spacer sequence and work in concert to generate a cleavage sitewithin the spacer sequence. Given a predetermined DNA locus or spacersequence, upstream and downstream half-sites can be identified using anumber of programs known in the art (Kornel Labun; Tessa G. Montague;James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016). CHOPCHOP v2: aweb tool for the next generation of CRISPR genome engineering. NucleicAcids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz;James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: aCRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res.42. W401-W407). It is also understood that a TALEN recognition sequencecan be defined as the DNA binding sequence (i.e., half-site) of a singleTALEN protein or, alternatively, a DNA sequence comprising the upstreamhalf-site, the spacer sequence, and the downstream half-site.

As used herein, the term “compact TALEN” refers to an endonucleasecomprising a DNA-binding domain with one or more TAL domain repeatsfused in any orientation to any portion of the I-TevI homingendonuclease or any of the endonucleases listed in Table 2 in U.S.Application No. 20130117869 (which is incorporated by reference in itsentirety), including but not limited to MmeI, EndA, End1, I-BasI,I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs donot require dimerization for DNA processing activity, alleviating theneed for dual target sites with intervening DNA spacers. In someembodiments, the compact TALEN comprises 16-22 TAL domain repeats.

As used herein, the term “megaTAL” refers to a single-chain endonucleasecomprising a transcription activator-like effector (TALE) DNA bindingdomain with an engineered, sequence-specific homing endonuclease.

As used herein, the terms “zinc finger nuclease” or “ZFN” refers to achimeric protein comprising a zinc finger DNA-binding domain fused to anuclease domain from an endonuclease or exonuclease, including but notlimited to a restriction endonuclease, homing endonuclease, S1 nuclease,mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeastHO endonuclease. Nuclease domains useful for the design of zinc fingernucleases include those from a Type IIs restriction endonuclease,including but not limited to FokI, FoM, and StsI restriction enzyme.Additional Type IIs restriction endonucleases are described inInternational Publication No. WO 2007/014275, which is incorporated byreference in its entirety. The structure of a zinc finger domain isstabilized through coordination of a zinc ion. DNA binding proteinscomprising one or more zinc finger domains bind DNA in asequence-specific manner. The zinc finger domain can be a nativesequence or can be redesigned through rational or experimental means toproduce a protein which binds to a pre-determined DNA sequence ˜18basepairs in length, comprising a pair of nine basepair half-sitesseparated by 2-10 basepairs. See, for example, U.S. Pat. Nos. 5,789,538,5,925,523, 6,007,988, 6,013,453, 6,200,759, and InternationalPublication Nos. WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO00/27878, WO 01/60970, WO 01/88197, and WO 02/099084, each of which isincorporated by reference in its entirety. By fusing this engineeredprotein domain to a nuclease domain, such as FokI nuclease, it ispossible to target DNA breaks with genome-level specificity. Theselection of target sites, zinc finger proteins and methods for designand construction of zinc finger nucleases are known to those of skill inthe art and are described in detail in U.S. Publications Nos.20030232410, 20050208489, 2005064474, 20050026157, 20060188987 andInternational Publication No. WO 07/014275, each of which isincorporated by reference in its entirety. In the case of a zinc finger,the DNA binding domains typically recognize an 18-bp recognitionsequence comprising a pair of nine basepair “half-sites” separated by a2-10 basepair “spacer sequence”, and cleavage by the nuclease creates ablunt end or a 5′ overhang of variable length (frequently fourbasepairs). It is understood that the term “zinc finger nuclease” canrefer to a single zinc finger protein or, alternatively, a pair of zincfinger proteins (i.e., a left ZFN protein and a right ZFN protein) thatbind to the upstream and downstream half-sites adjacent to the zincfinger nuclease spacer sequence and work in concert to generate acleavage site within the spacer sequence. Given a predetermined DNAlocus or spacer sequence, upstream and downstream half-sites can beidentified using a number of programs known in the art (Mandell J G,Barbas C F 3rd. Zinc Finger Tools: custom DNA-binding domains fortranscription factors and nucleases. Nucleic Acids Res. 2006 Jul. 1; 34(Web Server issue):W516-23). It is also understood that a zinc fingernuclease recognition sequence can be defined as the DNA binding sequence(i.e., half-site) of a single zinc finger nuclease protein or,alternatively, a DNA sequence comprising the upstream half-site, thespacer sequence, and the downstream half-site.

As used herein, the terms “CRISPR nuclease” or “CRISPR system nuclease”refers to a CRISPR (clustered regularly interspaced short palindromicrepeats)-associated (Cas) endonuclease or a variant thereof, such asCas9, that associates with a guide RNA that directs nucleic acidcleavage by the associated endonuclease by hybridizing to a recognitionsite in a polynucleotide. In certain embodiments, the CRISPR nuclease isa class 2 CRISPR enzyme. In some of these embodiments, the CRISPRnuclease is a class 2, type II enzyme, such as Cas9. In otherembodiments, the CRISPR nuclease is a class 2, type V enzyme, such asCpf1. The guide RNA comprises a direct repeat and a guide sequence(often referred to as a spacer in the context of an endogenous CRISPRsystem), which is complementary to the target recognition site. Incertain embodiments, the CRISPR system further comprises a tracrRNA(trans-activating CRISPR RNA) that is complementary (fully or partially)to the direct repeat sequence (sometimes referred to as a tracr-matesequence) present on the guide RNA. In particular embodiments, theCRISPR nuclease can be mutated with respect to a corresponding wild-typeenzyme such that the enzyme lacks the ability to cleave one strand of atarget polynucleotide, functioning as a nickase, cleaving only a singlestrand of the target DNA. Non-limiting examples of CRISPR enzymes thatfunction as a nickase include Cas9 enzymes with a D10 A mutation withinthe RuvC I catalytic domain, or with a H840 A, N854 A, or N863 Amutation. Given a predetermined DNA locus, recognition sequences can beidentified using a number of programs known in the art (Kornel Labun;Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen.(2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genomeengineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G.Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen.(2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.Nucleic Acids Res. 42. W401-W407).

As used herein, a “template nucleic acid” refers to a nucleic acid(i.e., a polynucleotide) that is desired to be inserted into a cleavagesite within a cell's genome.

As used herein, the terms “recombinant” or “engineered,” with respect toa protein, means having an altered amino acid sequence as a result ofthe application of genetic engineering techniques to nucleic acids thatencode the protein and cells or organisms that express the protein. Withrespect to a nucleic acid, the term “recombinant” or “engineered” meanshaving an altered nucleic acid sequence as a result of the applicationof genetic engineering techniques. Genetic engineering techniquesinclude, but are not limited to, PCR and DNA cloning technologies;transfection, transformation, and other gene transfer technologies;homologous recombination; site-directed mutagenesis; and gene fusion. Inaccordance with this definition, a protein having an amino acid sequenceidentical to a naturally-occurring protein, but produced by cloning andexpression in a heterologous host, is not considered recombinant orengineered.

As used herein, the term “wild-type” refers to the most common naturallyoccurring allele (i.e., polynucleotide sequence) in the allelepopulation of the same type of gene, wherein a polypeptide encoded bythe wild-type allele has its original functions. The term “wild-type”also refers to a polypeptide encoded by a wild-type allele. Wild-typealleles (i.e., polynucleotides) and polypeptides are distinguishablefrom mutant or variant alleles and polypeptides, which comprise one ormore mutations and/or substitutions relative to the wild-typesequence(s). Whereas a wild-type allele or polypeptide can confer anormal phenotype in an organism, a mutant or variant allele orpolypeptide can, in some instances, confer an altered phenotype.Wild-type nucleases are distinguishable from recombinant ornon-naturally-occurring nucleases. The term “wild-type” can also referto a cell, an organism, and/or a subject which possesses a wild-typeallele of a particular gene, or a cell, an organism, and/or a subjectused for comparative purposes.

As used herein, the term “genetically-modified” refers to a cell ororganism in which, or in an ancestor of which, a genomic DNA sequencehas been deliberately modified by recombinant technology. As usedherein, the term “genetically-modified” encompasses the term“transgenic.”

As used herein with respect to recombinant proteins, the term“modification” means any insertion, deletion, or substitution of anamino acid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the terms “recognition sequence” or “recognition site”refers to a DNA sequence that is bound and cleaved by a nuclease. In thecase of a meganuclease, a recognition sequence comprises a pair ofinverted, 9 basepair “half sites” which are separated by four basepairs.In the case of a single-chain meganuclease, the N-terminal domain of theprotein contacts a first half-site and the C-terminal domain of theprotein contacts a second half-site. Cleavage by a meganuclease producesfour basepair 3′ overhangs. “Overhangs,” or “sticky ends” are short,single-stranded DNA segments that can be produced by endonucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesand single-chain meganucleases derived from I-CreI, the overhangcomprises bases 10-13 of the 22 basepair recognition sequence. In thecase of a compact TALEN, the recognition sequence comprises a firstCNNNGN sequence that is recognized by the I-TevI domain, followed by anon-specific spacer 4-16 basepairs in length, followed by a secondsequence 16-22 bp in length that is recognized by the TAL-effectordomain (this sequence typically has a 5′ T base). Cleavage by a compactTALEN produces two basepair 3′ overhangs. In the case of a CRISPRnuclease, the recognition sequence is the sequence, typically 16-24basepairs, to which the guide RNA binds to direct cleavage. Fullcomplementarity between the guide sequence and the recognition sequenceis not necessarily required to effect cleavage. Cleavage by a CRISPRnuclease can produce blunt ends (such as by a class 2, type II CRISPRnuclease) or overhanging ends (such as by a class 2, type V CRISPRnuclease), depending on the CRISPR nuclease. In those embodimentswherein a CpfI CRISPR nuclease is utilized, cleavage by the CRISPRcomplex comprising the same will result in 5′ overhangs and in certainembodiments, 5 nucleotide 5′ overhangs. Each CRISPR nuclease enzyme alsorequires the recognition of a PAM (protospacer adjacent motif) sequencethat is near the recognition sequence complementary to the guide RNA.The precise sequence, length requirements for the PAM, and distance fromthe target sequence differ depending on the CRISPR nuclease enzyme, butPAMs are typically 2-5 base pair sequences adjacent to thetarget/recognition sequence. PAM sequences for particular CRISPRnuclease enzymes are known in the art (see, for example, U.S. Pat. No.8,697,359 and U.S. Publication No. 20160208243, each of which isincorporated by reference in its entirety) and PAM sequences for novelor engineered CRISPR nuclease enzymes can be identified using methodsknown in the art, such as a PAM depletion assay (see, for example,Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated hereinin its entirety). In the case of a zinc finger, the DNA binding domainstypically recognize an 18-bp recognition sequence comprising a pair ofnine basepair “half-sites” separated by 2-10 basepairs and cleavage bythe nuclease creates a blunt end or a 5′ overhang of variable length(frequently four basepairs).

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a nuclease.

As used herein, the terms “DNA-binding affinity” or “binding affinity”means the tendency of a nuclease to non-covalently associate with areference DNA molecule (e.g., a recognition sequence or an arbitrarysequence). Binding affinity is measured by a dissociation constant, Kd.As used herein, a nuclease has “altered” binding affinity if the Kd ofthe nuclease for a reference recognition sequence is increased ordecreased by a statistically significant percent change relative to areference nuclease.

As used herein, the term “specificity” means the ability of a nucleaseto bind and cleave double-stranded DNA molecules only at a particularsequence of base pairs referred to as the recognition sequence, or onlyat a particular set of recognition sequences. The set of recognitionsequences will share certain conserved positions or sequence motifs butmay be degenerate at one or more positions. A highly-specific nucleaseis capable of cleaving only one or a very few recognition sequences.Specificity can be determined by any method known in the art.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.In some instances, cleavage at a target recognition sequence results inNHEJ at a target recognition site. Nuclease-induced cleavage of a targetsite in the coding sequence of a gene followed by DNA repair by NHEJ canintroduce mutations into the coding sequence, such as frameshiftmutations, that disrupt gene function. Thus, engineered nucleases can beused to effectively knock-out a gene in a population of cells.

As used herein, the term “disrupted” or “disrupts” or “disruptsexpression” or “disrupting a target sequence” refers to the introductionof a mutation (e.g., frameshift mutation) that interferes with the genefunction and prevents expression and/or function of thepolypeptide/expression product encoded thereby. For example,nuclease-mediated disruption of a gene can result in the expression of atruncated protein and/or expression of a protein that does not retainits wild-type function. Additionally, introduction of a template nucleicacid into a gene can result in no expression of an encoded protein,expression of a truncated protein, and/or expression of a protein thatdoes not retain its wild-type function.

As used herein, the term “chimeric antigen receptor” or “CAR” refers toan engineered receptor that confers or grafts specificity for an antigenonto an immune effector cell (e.g., a human T cell). A chimeric antigenreceptor comprises at least an extracellular ligand-binding domain ormoiety, a transmembrane domain, and an intracellular domain thatcomprises one or more signaling domains and/or co-stimulatory domains.

In some embodiments, the extracellular ligand-binding domain or moietyis an antibody, or antibody fragment. In this context, the term“antibody fragment” can refer to at least one portion of an antibody,that retains the ability to specifically interact with (e.g., bybinding, steric hindrance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies).

In some embodiments, the extracellular ligand-binding domain or moietyis in the form of a single-chain variable fragment (scFv) derived from amonoclonal antibody, which provides specificity for a particular epitopeor antigen (e.g., an epitope or antigen preferentially present on thesurface of a cell, such as a cancer cell or other disease-causing cellor particle). In some embodiments, the scFv is attached via a linkersequence. In some embodiments, the scFv is murine, humanized, or fullyhuman.

The extracellular ligand-binding domain of a chimeric antigen receptorcan also comprise an autoantigen (see, Payne et al. (2016), Science 353(6295): 179-184), that can be recognized by autoantigen-specific B cellreceptors on B lymphocytes, thus directing T cells to specificallytarget and kill autoreactive B lymphocytes in antibody-mediatedautoimmune diseases. Such CARs can be referred to as chimericautoantibody receptors (CAARs), and their use is encompassed by theinvention. The extracellular ligand-binding domain of a chimeric antigenreceptor can also comprise a naturally-occurring ligand for an antigenof interest, or a fragment of a naturally-occurring ligand which retainsthe ability to bind the antigen of interest.

The intracellular stimulatory domain can include one or more cytoplasmicsignaling domains that transmit an activation signal to the T cellfollowing antigen binding. Such cytoplasmic signaling domains caninclude, without limitation, a CD3 zeta signaling domain.

The intracellular stimulatory domain can also include one or moreintracellular co-stimulatory domains that transmit a proliferativeand/or cell-survival signal after ligand binding. Such intracellularco-stimulatory domains can be any of those known in the art and caninclude, without limitation, those co-stimulatory domains disclosed inWO 2018/067697 including, for example, Novel 6. Further examples ofco-stimulatory domains can include 4-1BB (CD137), CD27, CD28, CD8, OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically bindswith CD83, or any combination thereof.

A chimeric antigen receptor further includes additional structuralelements, including a transmembrane domain that is attached to theextracellular ligand-binding domain via a hinge or spacer sequence. Thetransmembrane domain can be derived from any membrane-bound ortransmembrane protein. For example, the transmembrane polypeptide can bea subunit of the T-cell receptor (e.g., an α, β, γ or ζ, polypeptideconstituting CD3 complex), IL2 receptor p55 (a chain), p75 (β chain) orγ chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CDproteins such as the CD8 alpha chain. In certain examples, thetransmembrane domain is a CD8 alpha domain. Alternatively, thetransmembrane domain can be synthetic and can comprise predominantlyhydrophobic residues such as leucine and valine.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcyRllla receptoror IgGl. In certain examples, the hinge region can be a CD8 alphadomain.

As used herein, the terms “exogenous T cell receptor” or “exogenous TCR”refer to a TCR whose sequence is introduced into the genome of an immunecell (e.g., a human T cell) that may or may not endogenously express theTCR. Expression of an exogenous TCR on an immune cell can conferspecificity for a specific epitope or antigen (e.g., an epitope orantigen preferentially present on the surface of a cancer cell or otherdisease-causing cell or particle). Such exogenous T cell receptors cancomprise alpha and beta chains or, alternatively, may comprise gamma anddelta chains. Exogenous TCRs useful in the invention may havespecificity to any antigen or epitope of interest.

As used herein, the term “HLA class I histocompatibility antigen, alphachain E fusion protein” or “HLA-E fusion protein” refers to a proteincomprising an HLA-E protein fused to at least one additional proteinthat enables expression of the HLA-E protein on the cell-surface. HLA-Eproteins can include, for example, an HLA-E-01:01 or HLA-E-01:03 protein(e.g., SEQ ID NO: 118). An HLA-E fusion protein can comprise, forexample, an HLA-E protein fused to a beta-2 microglobulin protein (e.g.,SEQ ID NO: 119) that enables expression of the HLA-E protein on thecell-surface. In further examples, the HLA-E fusion protein can comprisean HLA-E protein fused to both a beta-2 microglobulin protein and anadditional protein that is loaded into the HLA-E protein forpresentation such as, for example, an HLA-G leader peptide (e.g., SEQ IDNO: 120) and others known in the art. The proteins of the HLA-E fusionprotein can be fused by polypeptide linkers such as, for example, alinker comprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) or SEQ ID NO:122 (i.e., a (GGGGS)4 linker).

As used herein, the term “reduced expression” in reference to a targetprotein (i.e., an endogenously expressed protein) refers to anyreduction in the expression of the endogenous protein by agenetically-modified cell when compared to a control cell. The termreduced can also refer to a reduction in the percentage of cells in apopulation of cells that express wild-type levels of an endogenousprotein targeted by a shRNAmiR of the disclosure when compared to apopulation of control cells. Such a reduction in the percentage of cellsin a population that fully express the targeted endogenous protein maybe up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or up to 100%. It is understood in the context of thisdisclosure that the term “reduced” encompasses a partial or incompleteknockdown of a target or endogenous protein, and is distinguished from acomplete knockdown, such as that achieved by gene inactivation by anuclease.

As used herein, the term with respect to both amino acid sequences andnucleic acid sequences, the terms “percent identity,” “sequenceidentity,” “percentage similarity,” “sequence similarity” and the likerefer to a measure of the degree of similarity of two sequences basedupon an alignment of the sequences that maximizes similarity betweenaligned amino acid residues or nucleotides, and which is a function ofthe number of identical or similar residues or nucleotides, the numberof total residues or nucleotides, and the presence and length of gaps inthe sequence alignment. A variety of algorithms and computer programsare available for determining sequence similarity using standardparameters. As used herein, sequence similarity is measured using theBLASTp program for amino acid sequences and the BLASTn program fornucleic acid sequences, both of which are available through the NationalCenter for Biotechnology Information (see, the website atncbi.nlm.nih.gov), and are described in, for example, Altschul et al.(1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet.3:266-272; Madden et al. (1996), Meth. Enzymol. 266:131-141; Altschul etal. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J.Comput. Biol. 7(1-2):203-14. As used herein, percent similarity of twoamino acid sequences is the score based upon the following parametersfor the BLASTp algorithm: word size=3; gap opening penalty=−11; gapextension penalty=−1; and scoring matrix=BLOSUM62. As used herein,percent similarity of two nucleic acid sequences is the score based uponthe following parameters for the BLASTn algorithm: word size=11; gapopening penalty=−5; gap extension penalty=−2; match reward=1; andmismatch penalty=−3.

As used herein, the term “corresponding to” with respect tomodifications of two proteins or amino acid sequences is used toindicate that a specified modification in the first protein is asubstitution of the same amino acid residue as in the modification inthe second protein, and that the amino acid position of the modificationin the first protein corresponds to or aligns with the amino acidposition of the modification in the second protein when the two proteinsare subjected to standard sequence alignments (e.g., using the BLASTpprogram). Thus, the modification of residue “X” to amino acid “A” in thefirst protein will correspond to the modification of residue “Y” toamino acid “A” in the second protein if residues X and Y correspond toeach other in a sequence alignment and despite the fact that X and Y maybe different numbers.

As used herein, the term “T cell receptor alpha gene” or “TCR alphagene” refer to the locus in a T cell which encodes the T cell receptoralpha subunit. The T cell receptor alpha gene can refer to NCBI Gene IDnumber 6955, before or after rearrangement. Following rearrangement, theT cell receptor alpha gene comprises an endogenous promoter, rearrangedV and J segments, the endogenous splice donor site, an intron, theendogenous splice acceptor site, and the T cell receptor alpha constantregion locus, which comprises the subunit coding exons.

As used herein, the term “T cell receptor alpha constant region” or “TCRalpha constant region” refers to the coding sequence of the T cellreceptor alpha gene. The TCR alpha constant region includes thewild-type sequence, and functional variants thereof, identified by NCBIGene ID NO. 28755.

As used herein, the term “T cell receptor beta gene” or “TCR beta gene”refers to the locus in a T cell which encodes the T cell receptor betasubunit. The T cell receptor beta gene can refer to NCBI Gene ID number6957.

As used herein, the term “recombinant DNA construct,” “recombinantconstruct,” “cassette,” “expression cassette,” “expression construct,”“chimeric construct,” “construct,” and “recombinant DNA fragment” areused interchangeably herein and are single or double-strandedpolynucleotides. A recombinant construct comprises an artificialcombination of nucleic acid fragments, including, without limitation,regulatory and coding sequences that are not found together in nature.For example, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource and arranged in a manner different than that found in nature.Such a construct may be used by itself or may be used in conjunctionwith a vector.

As used herein, the term “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used, then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant AAV vectors, or anyother vector known in the art suitable for delivering a gene to a targetcell. The skilled artisan is well aware of the genetic elements thatmust be present on the vector in order to successfully transform, selectand propagate host cells comprising any of the isolated nucleotides ornucleic acid sequences of the invention.

As used herein, a “vector” can also refer to a viral vector (i.e., arecombinant virus). Viral vectors can include, without limitation,retroviral vectors (i.e., recombinant retroviruses), lentiviral vectors(i.e., recombinant lentiviruses), adenoviral vectors (i.e., recombinantadenoviruses), and adeno-associated viral vectors (AAV) (i.e.,recombinant AAVs).

As used herein, the term “immune cell” refers to any cell that is partof the immune system (innate and/or adaptive) and is of hematopoieticorigin. Non-limiting examples of immune cells include lymphocytes, Bcells, T cells, monocytes, macrophages, dendritic cells, granulocytes,megakaryocytes, monocytes, macrophages, natural killer cells,myeloid-derived suppressor cells, innate lymphoid cells, platelets, redblood cells, thymocytes, leukocytes, neutrophils, mast cells,eosinophils, basophils, and granulocytes.

As used herein, a “human T cell” or “T cell” or “isolated human T cell”refers to a T cell isolated from a donor, particularly a human donor. Tcells, and cells derived therefrom, include isolated T cells that havenot been passaged in culture, T cells that have been passaged andmaintained under cell culture conditions without immortalization, and Tcells that have been immortalized and can be maintained under cellculture conditions indefinitely.

As used herein, a “human NK cell” or “NK cell” refers to a NK cellisolated from a donor, particularly a human donor. NK cells, and cellsderived therefrom, include isolated NK cells that have not been passagedin culture, NK cells that have been passaged and maintained under cellculture conditions without immortalization, and NK cells that have beenimmortalized and can be maintained under cell culture conditionsindefinitely.

As used herein, a “human B cell” or “B cell” refers to a B cell isolatedfrom a donor, particularly a human donor. B cells, and cells derivedtherefrom, include isolated T cells that have not been passaged inculture, B cells that have been passaged and maintained under cellculture conditions without immortalization, and B cells that have beenimmortalized and can be maintained under cell culture conditionsindefinitely.

As used herein, the term “a control” or “a control cell” refers to acell that provides a reference point for measuring changes in genotypeor phenotype of a genetically-modified cell. A control cell maycomprise, for example: (a) a wild-type cell, i.e., of the same genotypeas the starting material for the genetic alteration which resulted inthe genetically-modified cell; (b) a cell of the same genotype as thegenetically-modified cell but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest); or, (c) a cell genetically identical to thegenetically-modified cell but which is not exposed to conditions orstimuli or further genetic modifications that would induce expression ofaltered genotype or phenotype.

As used herein, the terms “treatment” or “treating a subject” refers tothe administration of a genetically-modified immune cell or populationof genetically-modified immune cells of the invention to a subjecthaving a disease. For example, the subject can have a disease such ascancer, and treatment can represent immunotherapy for the treatment ofthe disease. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, decreasing the rate of disease progression,amelioration or palliation of the disease state, and remission orimproved prognosis. In some aspects, a genetically-modified immune cellor population of genetically-modified immune cells described herein isadministered during treatment in the form of a pharmaceuticalcomposition of the invention.

The term “effective amount” or “therapeutically effective amount” refersto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results. The therapeutically effective amount will varydepending on the formulation or composition used, the disease and itsseverity and the age, weight, physical condition and responsiveness ofthe subject to be treated. In specific embodiments, an effective amountof a genetically-modified immune cell or population ofgenetically-modified immune cells of the invention, or pharmaceuticalcompositions disclosed herein, reduces at least one symptom of a diseasein a subject. In those embodiments wherein the disease is a cancer, aneffective amount of the pharmaceutical compositions disclosed hereinreduces the level of proliferation or metastasis of cancer, causes apartial or full response or remission of cancer, or reduces at least onesymptom of cancer in a subject.

As used herein, the term “cancer” should be understood to encompass anyneoplastic disease (whether invasive or metastatic) which ischaracterized by abnormal and uncontrolled cell division causingmalignant growth or tumor.

As used herein, the term “carcinoma” refers to a malignant growth madeup of epithelial cells.

As used herein, the term “leukemia” refers to malignancies of thehematopoietic organs/systems and is generally characterized by anabnormal proliferation and development of leukocytes and theirprecursors in the blood and bone marrow.

As used herein, the term “sarcoma” refers to a tumor which is made up ofa substance like the embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillary,heterogeneous, or homogeneous substance.

As used herein, the term “melanoma” refers to a tumor arising from themelanocytic system of the skin and other organs.

As used herein, the term “lymphoma” refers to a group of blood celltumors that develop from lymphocytes.

As used herein, the term “blastoma” refers to a type of cancer that iscaused by malignancies in precursor cells or blasts (immature orembryonic tissue).

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 can takethe values 0, 1 or 2 if the variable is inherently discrete, and cantake the values 0.0, 0.1, 0.01, 0.001, or any other real values and ifthe variable is inherently continuous.

2.1 Principle of the Invention

The present invention is based, in part, on the discovery thatmicroRNA-adapted shRNA (shRNAmiR) molecules can be used to generategenetically-modified immune cells having a stable reduction inexpression of an endogenous protein. It is demonstrated herein that theinsertion of a nucleic acid sequence encoding a shRNAmiR into the genomeof a T cell provides stable knockdown of a spectrum of endogenousproteins including, for example, the stable and effective knockdown ofbeta-2 microglobulin (B2M), CS1, transforming growth factor betareceptor II (TGFBR2), Cbl proto-oncogene B (CBL-B), deoxycytidine kinase(DCK), glucocorticoid (GR), and cluster of differentiation 52 (CD52).Knockdown of endogenous proteins can confer properties that, in someinstances, can be advantageous compared to knockout by geneinactivation. For example, B2M knockdown by shRNAmiR produces CAR Tcells that are less allogeneic and less susceptible to natural killer(NK) cell killing than CAR T cells exhibiting a complete knockout ofB2M. Further, it is demonstrated that the incorporation of a shRNAmiRmolecule into the genome of an immune cell solves the stability andtoxicity problems observed with the insertion of a cassette encoding anshRNA molecule.

Given the demonstration that shRNAmiR molecules can be used to reducethe expression of multiple endogenous proteins, the presently disclosedcompositions and methods can be used to stably knockdown the expressionat various degrees of not only the B2M protein, but any endogenousprotein of interest, within an immune cell.

2.2 MicroRNA-Adapted shRNA (shRNAmiR)

RNA interference (RNAi) or RNA silencing refers to a process by whichgene expression is negatively regulated by non-coding RNAs such asmicroRNAs. The negative regulation can result from one or more of threepossible mechanisms: (1) by repressing the translation of target mRNAs,(2) through deadenylation and destabilization of transcripts, and (3)through cleavage and degradation of mRNAs. RNAi is normally triggered bydouble-stranded RNA (dsRNA) or endogenous microRNA precursors(pri-miRNAs/pre-miRNAs).

The production of endogenous microRNA molecules begins with thetranscription of a primary miRNA (pri-mRNA) from an RNA polymerase II(Pol II) promoter. Each pri-miRNA can contain from one to sixpre-miRNAs. Pre-miRNAs are hairpin loop structures composed of about 70nucleotides, with each hairpin being flanked by sequences necessary forefficient processing. The enzyme Drosha liberates hairpins from thepri-miRNAs by cleaving RNA about 11 nucleotides from the hairpin base.Pre-miRNAs that are generated by Drosha cleavage comprise a 2 nucleotideoverhang at the 3′ end. This 2 nucleotide overhang is bound by theExportin-5 protein, which mediates export of the pre-miRNA from thenucleus into the cytoplasm. In the cytoplasm, pre-miRNA hairpins arecleaved by Dicer through interactions with the 5′ and 3′ ends of thehairpin. Dicer cleaves the pre-miRNA hairpin in the loop region toproduce an imperfect miRNA:miRNA duplex, which is about 22 nucleotidesin length. A single strand of the miRNA:miRNA duplex (mature miRNA) isincorporated into the RNA-induced silencing complex (RISC) where themiRNA and its mRNA target interact.

Since its discovery, RNAi has emerged as a powerful genetic tool forsuppressing gene expression in mammalian cells. Gene knockdown can beachieved by expression of synthetic short hairpin RNAs (shRNAs) thatmimic pre-miRNAs and are processed by Dicer and fed into the RISC.However, as described herein, shRNAs may not allow for prolongedreduction of protein expression in immune cells. In contrast, expressionof the microRNA-adapted shRNA (shRNAmiR) molecules of the presentinvention result in persistent reduction of protein expression andreduced toxicity effects. The shRNAmiR molecules mimic pri-miRNAmolecules in that they comprise a hairpin flanked by sequences necessaryfor efficient processing, and can be processed by the Drosha enzyme intopri-miRNAs, exported into the cytoplasm, and cleaved by Dicer, afterwhich the mature miRNA can enter the RISC.

The present invention provides genetically-modified immune cellsexpressing a shRNAmiR molecule that reduces the abundance of anendogenous protein.

The shRNAmiR molecule can comprise a microRNA scaffold in that thestructure of the shRNAmiR molecule can mimic that of anaturally-occurring microRNA (or pri-miRNA or pre-miRNA) or a variantthereof. Sequences of microRNAs (and pri-miRNAs and pre-miRNAs) areknown in the art. Non-limiting examples of suitable miR scaffolds forthe presently disclosed shRNAmiRs include miR-E, miR-30 (e.g., miR-30a),miR-15, miR-16, miR-155, miR-22, miR-103, and miR-107. In particularembodiments, the shRNAmiR used in the presently disclosed compositionsand methods comprises a mir-E scaffold. The mir-E scaffold is asynthetically-derived variant of miR-30a and its genesis is described inInternational Publication No. WO 2014/117050, which is incorporated byreference in its entirety.

The presently disclosed shRNAmiR molecules can comprise the followingdomains in the 5′ to 3′ direction: (a) a 5′ miR scaffold domain; (b) a5′ miR basal stem domain; (c) a passenger strand; (d) a miR loop domain;(e) a guide strand; (f) a 3′ miR basal stem domain; and (g) a 3′ miRscaffold domain. The miR scaffold domains and basal stem domains flankthe miRNA stem-loop and are referred to herein as microRNA flankingsequences that comprise the microRNA processing elements (the minimalnucleic acid sequences which contribute to the production of maturemicroRNA from primary microRNA or precursor microRNA). Often theseelements are located within a 40 nucleotide sequence that flanks amicroRNA stem-loop structure. In some instances, the microRNA processingelements are found within a stretch of nucleotide sequences of between 5and 4,000 nucleotides in length that flank a microRNA stem-loopstructure.

In some embodiments, the miRNA flanking sequences are about 3 to about4,000 nt in length and can be present on either or both the 5′ and 3′ends of the shRNAmiR molecule. In other embodiments, the minimal lengthof the microRNA flanking sequence of the shRNAmiR molecule is about 10,about 20, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 125, about 126, about 127, about 128, about129, about 130, about 131, about 132, about 133, about 134, about 135,about 136, about 137, about 138, about 139, about 140, about 150, about200, and any integer therein between. In other embodiments the maximallength of the microRNA flanking sequence of the shRNAmiR molecule isabout 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000,about 3,100, about 3,200, about 3,300, about 3,400, about 3,500, about3,600, about 3,700, about 3,800, about 3,900, about 4,000, and anyinteger therein between.

The microRNA flanking sequences may be native microRNA flankingsequences or artificial microRNA flanking sequences. A native microRNAflanking sequence is a nucleotide sequence that is ordinarily comprisedwithin naturally existing systems with microRNA sequences (i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo). Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking microRNAsequences in naturally existing systems. The artificial microRNAflanking sequences may be flanking sequences found naturally in thecontext of other microRNA sequences. Alternatively, they may be composedof minimal microRNA processing elements which are found within naturallyoccurring flanking sequences and inserted into other random nucleic acidsequences that do not naturally occur as flanking sequences or onlypartially occur as natural flanking sequences.

In some embodiments, the 5′ miR scaffold domain is about 10 to about 150nucleotides in length, including but not limited to about 10, about 20,about 30, about 40, about 50, about 60, about 70, about 80, about 90,about 100, about 110, about 120, about 130, about 140, and about 150nucleotides long. In some of these embodiments, the 5′ miR scaffolddomain is about 111 nucleotides in length. The 5′ miR scaffold domainmay comprise a 3′ sequence that is a recognition sequence for a type IISrestriction enzyme. In some of these embodiments, the 5′ miR scaffolddomain comprises a Xhol recognition sequence on its 3′ end. Inparticular embodiments, the 5′ miR scaffold domain has at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or moresequence identity to the sequence set forth as SEQ ID NO: 1. In certainembodiments, the 5′ miR scaffold domain has the sequence set forth asSEQ ID NO: 1.

The 5′ miR basal stem domain of the shRNAmiR can be about 5 to about 30nucleotides in length in some embodiments, including but not limited toabout 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, and about 30 nucleotides long. In someof these embodiments, the 5′ miR basal stem domain is about 20nucleotides in length. In particular embodiments, the 5′ miR basal stemdomain has at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% or more sequence identity to the sequence set forthas SEQ ID NO: 2. In certain embodiments, the 5′ miR basal stem domainhas the sequence set forth as SEQ ID NO: 2.

The shRNAmiR molecules of the presently disclosed compositions andmethods comprise a stem-loop structure, wherein the stem is comprised ofthe hybridized passenger and guide strands and the loop issingle-stranded. The miR loop domain can be derived from anaturally-occurring pre-microRNA or pri-microRNA loop sequence or avariant thereof. In some embodiments, the miR loop domain has thesequence of a loop domain from any one of miR-30 (e.g., miR-30a),miR-15, miR-16, miR-155, miR-22, miR-103, and miR-107. In particularembodiments, the shRNAmiR comprises a miR-30a loop domain, the sequenceof which is set forth as SEQ ID NO: 3.

In certain embodiments, the miR loop domain is about 5 to about 30nucleotides in length, including but not limited to about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about28, about 29, and about 30 nucleotides long. In some of theseembodiments, the miR loop domain is about 15 nucleotides in length. Inparticular embodiments, the miR loop domain has at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% or more sequenceidentity to the sequence set forth as SEQ ID NO: 3. In certainembodiments, the miR loop domain has the sequence set forth as SEQ IDNO: 3.

The 3′ miR basal stem domain of the shRNAmiR can be about 5 to about 30nucleotides in length in some embodiments, including but not limited toabout 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, and about 30 nucleotides long. In someof these embodiments, the 3′ miR basal stem domain is about 18nucleotides in length. In particular embodiments, the 3′ miR basal stemdomain has at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% or more sequence identity to the sequence set forthas SEQ ID NO: 4. In certain embodiments, the 3′ miR basal stem domainhas the sequence set forth as SEQ ID NO: 4.

In some embodiments, the 3′ miR scaffold domain is about 50 to about 150nucleotides in length, including but not limited to about 10, about 20,about 30, about 40, about 50, about 60, about 70, about 80, about 90,about 100, about 110, about 120, about 130, about 140, or about 150nucleotides long. In some of these embodiments, the 3′ miR scaffolddomain is about 116 nucleotides in length. In particular embodiments,the 3′ miR scaffold domain has at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% or more sequence identity to thesequence set forth as SEQ ID NO: 5. In certain embodiments, the 3′ miRscaffold domain has the sequence set forth as SEQ ID NO: 5.

The guide strand of the shRNAmiR is the sequence that targets the mRNA,leading to reduction in abundance of the protein encoded by the mRNA.After the guide strand binds to its target mRNA, RISC either degradesthe target transcript and/or prevents the target transcript from beingloaded into the ribsome for translation. The guide strand is ofsufficient complementarity with the target mRNA in order to lead toreduced expression of the target mRNA. In some embodiments, the guidestrand is at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 98%, at least about 99% or 100% complementary to thetarget mRNA sequence. In certain embodiments, the guide strandhybridizes with the target mRNA within a coding sequence. The guidestrand can comprise 1, 2, 3, 4, 5, or more mismatching nucleotides withthe target mRNA sequence. In other embodiments, the guide strandhybridizes with the target mRNA in a non-coding region, such as a 5′ or3′ untranslated region (UTR). In some embodiments, the guide strand isabout 15 to about 25 nucleotides in length, including but not limited toabout 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, and about 25 nucleotides long. In some ofthese embodiments, the guide strand is about 22 nucleotides in length.In particular embodiments wherein the shRNA sequence from which theshRNAmiR is derived is less than 22 nucleotides in length, which is thelength of most naturally-occurring microRNAs, an additional nucleotideis added to the shRNA sequence and in certain embodiments, thisadditional nucleotide is one that is complementary with thecorresponding position within the target mRNA.

The passenger strand of the shRNAmiR is the sequence that is fully orpartially complementary with the guide strand sequence. In someembodiments, the passenger strand is about 15 to about 25 nucleotides inlength, including but not limited to about 15 to about 25 nucleotides inlength, including but not limited to about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, andabout 25 nucleotides long. In some of these embodiments, the passengerstrand is about 22 nucleotides in length. The passenger strand can be atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, atleast about 98%, at least about 99% or 100% complementary to the guidestrand sequence. The passenger strand can comprise 1, 2, 3, 4, 5, ormore mismatching nucleotides with the guide strand. In certainembodiments, however, the guide:passenger strand duplex does notcomprise any mismatching nucleotides. In general, guide/passenger strandsequences should be selected that do not form any secondary structureswithin themselves. Further, the use of guide/passenger strand sequencesthat target sites within an mRNA that comprise single-nucleotidepolymorphisms should be avoided. Guide/passenger strand sequences thatare specific for the target mRNA are preferred to avoid any off-targeteffects (i.e., reduction in expression of non-target mRNAs).

In order to aid in the selection of suitable shRNAmiR guide/passengerstrands, or sequences for other shRNAmiR domains, any program known inthe art that models the predicted secondary structure of a RNA moleculecan be used, including but not limited to Mfold, RNAfold, and UNAFold.Any program known in the art that can predict the efficiency of a shRNAor miRNA guide/passenger sequence to target a particular mRNA can beused to select suitable guide/passenger strand sequences, including butnot limited to those disclosed in Agarwal et al. (2015) eLife 4:e05005;and Knott et al. (2014) Mol Cell 56(6):796-807, each of which isincorporated herein in its entirety.

2.3 Genetically-Modified Immune Cells

The invention provides genetically-modified immune cells and populationsthereof and methods for producing the same. In some embodiments, thegenetically-modified immune cells of the presently disclosedcompositions and methods are human immune cells. In some embodiments,the immune cells are T cells, or cells derived therefrom. In otherembodiments, the immune cells are natural killer (NK) cells, or cellsderived therefrom. In still other embodiments, the immune cells are Bcells, or cells derived therefrom. In yet other embodiments, the immunecells are monocyte or macrophage cells or cells derived therefrom.

Immune cells (e.g., T cells) can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments of the present disclosure, any number of T cell lines, NKcell lines, B cell lines, monocyte cells lines, or macrophage cell linesavailable in the art may be used. In some embodiments of the presentdisclosure, immune cells (e.g., T cells) are obtained from a unit ofblood collected from a subject using any number of techniques known tothe skilled artisan. In one embodiment, cells from the circulating bloodof an individual are obtained by apheresis. Immune cells of theinvention can also be induced pluripotent stem cell (iPSC)-derived cellsthat have been differentiated into functional immune cells (e.g., Tcells, NK cell, B cells).

The genetically-modified immune cells of the presently disclosedcompositions and methods comprise in the cells' genome a nucleic acidsequence encoding a shRNAmiR, leading to the reduction of expression ofa target protein.

In some of those embodiments wherein the expression of an endogenousprotein is reduced by a shRNAmiR, the expression of the endogenousprotein is reduced by at least about 10%, about 20%, about 30%, about40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or up to about 99% comparedto a control cell (e.g., a cell not expressing a shRNAmiR). Any methodknown in the art can be used to determine the expression level of anendogenous protein targeted by a shRNAmiR, including but not limited to,ELISA, flow cytometry, Western blot, immunocytochemistry, andimmunoprecipitation.

Expressing a shRNAmiR by cells within a population can lead to areduction in the percentage of cells in the population of cells thatfully express the endogenous protein to which the shRNAmiR is targetedwhen compared to a population of control cells. Such a reduction may beup to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or up to 100% of cells in the population.

A nucleic acid sequence encoding a shRNAmiR can be present in the genomeof the genetically-modified immune cell, for example, in a cassette.Such cassettes can be inserted into the genome, for example, byintroducing a template nucleic acid of the invention either by randomintegration (e.g., lentiviral transduction) or by targeted insertioninto a selected site (e.g., by nuclease-mediated targeted insertion).Cassettes comprising the shRNAmiR-encoding sequence can include, forexample, nucleic acid sequences encoding additional proteins, such asthose described herein (e.g., CARs, exogenous TCRs, fusion proteins),and may also include control sequences such as promoters and terminationsequences. The nucleic acid sequence encoding the shRNAmiR can bepositioned at any number of locations within the cassette or templatenucleic acid that allow for expression of the shRNAmiR. In someexamples, a nucleic acid sequence encoding a shRNAmiR is positionedbetween the stop codon of another transgene (e.g., a nucleic acidsequence encoding a CAR, exogenous TCR, or fusion protein) and atermination signal. In other examples, another transgene present in thecassette or template nucleic acid (e.g., a nucleic acid sequenceencoding a CAR, exogenous TCR, or fusion protein) comprises an intronthat is positioned within the transgene sequence. Here, “positioned” isintended to mean that the intron sequence is inserted into the transgenesequence, such that the resulting sequence comprises a 5′ portion of thetransgene, the intron sequence, and a 3′ portion of the transgene. Insome such examples, the nucleic acid sequence encoding the shRNAmiR canbe positioned within such an intron. Here, “positioned” is intended tomean that the shRNAmiR-encoding sequence is inserted into the intronsequence, such that the resulting sequence comprises a 5′ portion of theintron sequence, the shRNAmiR-encoding sequence, and a 3′ portion of theintron sequence. In such cases, the shRNAmiR is expressed and the intronsequence is spliced out by the cell when the transgene is expressed.Introns that can be included in this manner can be naturally-occurringintrons or, alternatively, synthetic introns. A particular example of asynthetic intron useful for the invention is encoded by SEQ ID NO: 69.

In certain embodiments, the genetically-modified immune cell can furthercomprise in its genome a nucleic acid sequence encoding a CAR or anexogenous TCR. In some embodiments, the genetically-modified immune cellcan further comprise in its genome a nucleic acid sequence encoding anHLA-E fusion protein capable of being expressed on the immune cellsurface.

The CAR/TCR-encoding nucleic acid sequence and/or the nucleic acidsequence encoding the HLA-E fusion protein can be located within thesame gene as the shRNAmiR-encoding sequence. Alternatively, theCAR/TCR-encoding nucleic acid sequence and/or the nucleic acid sequenceencoding the HLA-E fusion protein can be located within a different geneas the shRNAmiR-encoding sequence.

Each of the coding sequences can be operably linked to differentpromoters. In other embodiments, the shRNAmiR-encoding sequence isoperably linked to the same promoter as the nucleic acid sequenceencoding the CAR or exogenous TCR and/or the nucleic acid sequenceencoding the HLA-E fusion protein. In some specific examples, where anucleic acid sequence encoding a shRNAmiR, a nucleic acid sequenceencoding a CAR or exogenous TCR, and a nucleic acid sequence encoding anHLA-E fusion protein are all located within the same gene, two of thenucleic acid sequences are operably linked to a first promoter, and thethird nucleic acid sequence is operably linked to a second promoter. Inother specific examples, where a nucleic acid sequence encoding ashRNAmiR, a nucleic acid sequence encoding a CAR or exogenous TCR, and anucleic acid sequence encoding an HLA-E fusion protein are all locatedwithin the same gene, all three of the nucleic acid sequences areoperably linked to the same promoter. In various embodiments, thenucleic acid sequences can be operably linked to an endogenous promoterfollowing insertion into the genome. In some such cases, the cassettesor template nucleic acids of the invention may not require an exogenouspromoter in order for the encoded sequences to be expressed. Further, insuch cases, the cassettes or template nucleic acids may compriseelements (e.g., splice acceptor sequences, 2 A or IRES sequences, andthe like) necessary for the nucleic acids to be operably linked to theendogenous promoter. In other embodiments, the cassettes or templatenucleic acids of the invention comprise one or more exogenous promotersthat are operably linked to the nucleic acid sequences and driveexpression of the shRNAmiR, CAR or exogenous TCR, and/or HLA-E fusionprotein.

Each of the coding sequences can be present in the genome in the sameorientation or in different orientations from each other. For example,one coding sequence can be on the plus strand of the double-stranded DNAand another coding sequence on the minus strand. In some embodiments,the shRNAmiR-encoding nucleic acid sequence is 3′ downstream of thenucleic acid sequence encoding the CAR or exogenous TCR and/or thenucleic acid sequence encoding the HLA-E fusion protein. In alternativeembodiments, the shRNAmiR-encoding sequence is 5′ upstream of theCAR/TCR-encoding sequence and/or the nucleic acid sequence encoding theHLA-E fusion protein.

In certain embodiments, nucleic acid sequences, such as those encoding aCAR or exogenous TCR, a shRNAmiR, and/or an HLA-E fusion protein, areoperably linked to the same promoter and are separated by any elementknown in the art to allow for the translation of two or more genes(i.e., cistrons) from the same nucleic acid molecule. Such elements caninclude, but are not limited to, an IRES element, a T2 A element, a P2 Aelement (e.g., P2 A/furin), an E2 A element, and an F2 A element.

In certain embodiments, the genetically-modified immune cell comprises anucleic acid sequence encoding a cell-surface protein that protects theimmune cell from NK cell killing. In some examples, the nucleic acidsequence encodes a non-classical MHC I protein. Non-classical MEW classI proteins can include, without limitation, HLA-E, HLA-F, HLA-G, andHLA-H. In particular examples, the nucleic acid sequence encodes anHLA-E protein. Examples of HLA-E proteins include, without limitation,an HLA-E-01:01 protein or an HLA-E-01:03 protein (e.g., SEQ ID NO: 118).In particular examples of the invention, the nucleic acid sequenceencodes a fusion protein comprising a non-classical MHC class I protein(e.g., HLA-E) and at least one additional protein that enablesexpression of the MHC class I protein on the cell-surface of the immunecell. The fusion protein can comprise, for example, an MHC class Iprotein (e.g., HLA-E) fused to a beta-2 microglobulin protein (e.g., SEQID NO: 119), that enables expression of the MHC class I protein on thecell-surface. In order to inhibit expression of endogenous beta-2microglobulin, and not expression of a fusion protein that comprisesbeta-2 microglobulin, the beta-2 microglobulin coding sequence in thefusion protein can be altered (e.g., codon optimized) such that theshRNAmiR does not have specificity for the altered sequence. In furtherexamples, the fusion protein can comprise a non-classical MHC class Iprotein (e.g., HLA-E) fused to both a beta-2 microglobulin protein andan additional protein that is presented extracellularly by thenon-classical MHC. Such additional proteins can include, for example, anHLA-G leader peptide (e.g., SEQ ID NO: 120). The individual proteins ofthe fusion protein can be fused by polypeptide linkers such as, forexample, a linker comprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) orSEQ ID NO: 122 (i.e., a (GGGGS)4 linker).

In specific embodiments, the fusion protein is an HLA-E fusion proteincomprising an HLA-E protein, a beta-2 microglobulin protein, and anHLA-G leader peptide. In some such embodiments, the HLA-E protein is anHLA-E-01:01 protein or an HLA-E-01:03 protein, and in particularembodiments, the HLA-E protein is an HLA-E-01:03 protein having an aminoacid sequence of SEQ ID NO: 118. In some such embodiments, the beta-2microglobulin protein has an amino acid sequence of SEQ ID NO: 119, andthe HLA-G leader peptide has an amino acid sequence of SEQ ID NO: 120.In further such embodiments, the HLA-E protein, the beta-2 microglobulinprotein, and the HLA-G leader protein are fused by polypeptide linkerscomprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) or SEQ ID NO: 122(i.e., a (GGGGS)4 linker). In a particular embodiment, the fusionprotein comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or up to atleast 99% sequence identity to SEQ ID NO: 66. In a specific embodiment,the fusion protein comprises an amino acid sequence of SEQ ID NO: 66.

In certain embodiments, the genetically-modified immune cells comprise anucleic acid sequence encoding a chimeric antigen receptor (CAR).Generally, a CAR of the present disclosure will comprise at least anextracellular domain, a transmembrane domain, and an intracellulardomain. In some embodiments, the extracellular domain comprises atarget-specific binding element otherwise referred to as anextracellular ligand-binding domain or moiety. In some embodiments, theintracellular domain, or cytoplasmic domain, comprises at least oneco-stimulatory domain and one or more signaling domains.

In some embodiments, a CAR useful in the invention comprises anextracellular ligand-binding domain. The choice of ligand-binding domaindepends upon the type and number of ligands that define the surface of atarget cell. For example, the ligand-binding domain may be chosen torecognize a ligand that acts as a cell surface marker on target cellsassociated with a particular disease state. Thus, some examples of cellsurface markers that may act as ligands for the ligand-binding domain ina CAR can include those associated with viruses, bacterial and parasiticinfections, autoimmune disease, and cancer cells. In some embodiments, aCAR is engineered to target a cancer-specific antigen of interest by wayof engineering a desired ligand-binding moiety that specifically bindsto an antigen on a cancer (i.e., tumor) cell. In the context of thepresent disclosure, “cancer antigen,” tumor antigen,” “cancer-specificantigen,” or “tumor-specific antigen” refer to antigens that are commonto specific hyperproliferative disorders such as cancer.

In some embodiments, the extracellular ligand-binding domain of the CARis specific for any antigen or epitope of interest, particularly anytumor antigen or epitope of interest. As non-limiting examples, in someembodiments the antigen of the target is a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30,CD40, CD79B, IL1RAP, glypican 3 (GPC3), CLL-1, disialoganglioside GD2,ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids,glioma-associated antigen, B-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostasespecific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA,surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1),MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor(IGFl)-1, IGF-II, IGFI receptor, mesothelin, a major histocompatibilitycomplex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4,ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) andextra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnCAl) and fibroblast associated protein (fap); a lineage-specific ortissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34,CD38, CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, amajor histocompatibility complex (MEW) molecule, BCMA (CD269, TNFRSF17), CS1, or a virus-specific surface antigen such as an HIV-specificantigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specificantigen, a HPV-specific antigen such as the E6 or E7 oncoproteins, aLasse Virus-specific antigen, an Influenza Virus-specific antigen, aswell as any derivate or variant of these surface markers.

In some examples, the extracellular ligand-binding domain or moiety isan antibody, or antibody fragment. An antibody fragment can, forexample, be at least one portion of an antibody, that retains theability to specifically interact with (e.g., by binding, sterichindrance, stabilizing/destabilizing, spatial distribution) an epitopeof an antigen. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv fragments, scFv antibody fragments,disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1domains, linear antibodies, single domain antibodies such as sdAb(either VL or VH), camelid VHH domains, multi-specific antibodies formedfrom antibody fragments such as a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region, and anisolated CDR or other epitope binding fragments of an antibody. Anantigen binding fragment can also be incorporated into single domainantibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger andHudson, Nature Biotechnology 23:1126-1136, 2005). Antigen bindingfragments can also be grafted into scaffolds based on polypeptides suchas a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, whichdescribes fibronectin polypeptide minibodies).

In some embodiments, the extracellular ligand-binding domain or moietyis in the form of a single-chain variable fragment (scFv) derived from amonoclonal antibody, which provides specificity for a particular epitopeor antigen (e.g., an epitope or antigen preferentially present on thesurface of a cell, such as a cancer cell or other disease-causing cellor particle). In some such embodiments, the scFv can comprise a heavychain variable (VH) domain and a light chain variable (VL) domain from amonoclonal antibody having specificity for an antigen. In someembodiments, the scFv is attached via a linker sequence. In someembodiments, the scFv is murine, humanized, or fully human.

The extracellular ligand-binding domain of a chimeric antigen receptorcan also comprise an autoantigen (see, Payne et al. (2016), Science 353(6295): 179-184), that can be recognized by autoantigen-specific B cellreceptors on B lymphocytes, thus directing T cells to specificallytarget and kill autoreactive B lymphocytes in antibody-mediatedautoimmune diseases. Such CARs can be referred to as chimericautoantibody receptors (CAARs), and their use is encompassed by theinvention.

In some embodiments, the extracellular domain of a chimeric antigenreceptor can comprise a naturally-occurring ligand for an antigen ofinterest, or a fragment of a naturally-occurring ligand which retainsthe ability to bind the antigen of interest.

A CAR can comprise a transmembrane domain which links the extracellularligand-binding domain with the intracellular signaling andco-stimulatory domains via a hinge region or spacer sequence. Thetransmembrane domain can be derived from any membrane-bound ortransmembrane protein. For example, the transmembrane polypeptide can bea subunit of the T-cell receptor (e.g., an α, β, γ or ζ, polypeptideconstituting CD3 complex), IL2 receptor p55 (a chain), p75 (β chain) orγ chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CDproteins such as the CD8 alpha chain. In certain examples, thetransmembrane domain is a CD8 alpha domain. Alternatively, thetransmembrane domain can be synthetic and can comprise predominantlyhydrophobic residues such as leucine and valine.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcyRllla receptoror IgGl. In certain examples, the hinge region can be a CD8 alphadomain.

Intracellular signaling domains of a CAR are responsible for activationof at least one of the normal effector functions of the cell in whichthe CAR has been placed and/or activation of proliferative and cellsurvival pathways. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. The intracellular stimulatory domain can include one or morecytoplasmic signaling domains that transmit an activation signal to theT cell following antigen binding. Such cytoplasmic signaling domains caninclude, without limitation, a CD3 zeta signaling domain.

The intracellular stimulatory domain can also include one or moreintracellular co-stimulatory domains that transmit a proliferativeand/or cell-survival signal after ligand binding. In some cases, theco-stimulatory domain can comprise one or more TRAF-binding domains.Such intracellular co-stimulatory domains can be any of those known inthe art and can include, without limitation, those co-stimulatorydomains disclosed in WO 2018/067697 including, for example, Novel 6(“N6”). Further examples of co-stimulatory domains can include 4-1BB(CD137), CD27, CD28, CD8, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, or any combination thereof. Ina particular embodiment, the co-stimulatory domain is an N6 domain. Inanother particular embodiment, the co-stimulatory domain is a 4-1BBco-stimulatory domain.

In other embodiments, the genetically-modified immune cell comprises anucleic acid sequence encoding an exogenous T cell receptor (TCR). Suchexogenous T cell receptors can comprise alpha and beta chains or,alternatively, may comprise gamma and delta chains. Exogenous TCRsuseful in the invention may have specificity to any antigen or epitopeof interest such as, without limitation, any antigen or epitopedisclosed herein.

In particular embodiments, the CAR or the exogenous TCR can be specificfor any type of cancer cell. Such cancers can include, withoutlimitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancersof B cell origin, breast cancer, gastric cancer, neuroblastoma,osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, andHodgkin lymphoma. In specific embodiments, cancers and disorders includebut are not limited to pre-B ALL (pediatric indication), adult ALL,mantle cell lymphoma, diffuse large B cell lymphoma, salvage postallogenic bone marrow transplantation, and the like. These cancers canbe treated using a combination of CARs that target, for example, CD19,CD20, CD22, and/or ROR1. In some non-limiting examples, agenetically-modified immune cell or population thereof of the presentdisclosure targets carcinomas, lymphomas, sarcomas, melanomas,blastomas, leukemias, and germ cell tumors, including but not limited tocancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer,renal cell carcinoma, liver cancer, gastric cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, breastcancer, lung cancer, cutaneous or intraocular malignant melanoma, renalcancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, non-Hodgkin lymphoma, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, solid tumors of childhood, lymphocytic lymphoma, cancer ofthe bladder, cancer of the kidney or ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, environmentally induced cancers including those induced byasbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphomas,acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoidleukemia, immunoblastic large cell lymphoma, acute lymphoblasticleukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-celllymphoma, and any combinations of said cancers. In certain embodiments,cancers of B-cell origin include, without limitation, B-lineage acutelymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-celllymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatricindication), mantle cell lymphoma, follicular lymphoma, marginal zonelymphoma, Burkitt's lymphoma, multiple myeloma, and B-cell non-Hodgkinlymphoma. In some examples, cancers can include, without limitation,cancers of B cell origin or multiple myeloma. In some examples, thecancer of B cell origin is acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), ornon-Hodgkin lymphoma (NHL). In some examples, the cancer of B cellorigin is mantle cell lymphoma (MCL) or diffuse large B cell lymphoma(DLBCL).

In some embodiments, genetically-modified immune cells of the inventioncomprise an inactivated TCR alpha gene and/or an inactivated TCR betagene. Inactivation of the TCR alpha gene and/or TCR beta gene togenerate the genetically-modified cells of the present invention occursin at least one or both alleles where the TCR alpha gene and/or TCR betagene is being expressed. Accordingly, inactivation of one or both genesprevents expression of the endogenous TCR alpha chain or the endogenousTCR beta chain protein. Expression of these proteins is required forassembly of the endogenous alpha/beta TCR on the cell surface. Thus,inactivation of the TCR alpha gene and/or the TCR beta gene results ingenetically-modified immune that have no detectable cell surfaceexpression of the endogenous alpha/beta TCR. The endogenous alpha/betaTCR incorporates CD3. Therefore, cells with an inactivated TCR alphagene and/or TCR beta chain can have no detectable cell surfaceexpression of CD3. In particular embodiments, the inactivated gene is aTCR alpha constant region (TRAC) gene.

In some examples, the TCR alpha gene, the TRAC gene, or the TCR betagene is inactivated by insertion of a template nucleic acid into acleavage site in the gene. Insertion of the template nucleic aciddisrupts expression of the endogenous TCR alpha chain or TCR beta chainand, therefore, prevents assembly of an endogenous alpha/beta TCR on theT cell surface. In some examples, the template nucleic acid is insertedinto the TRAC gene. In a particular example, a template nucleic acid isinserted into the TRAC gene at an engineered meganuclease recognitionsequence comprising SEQ ID NO: 58 (i.e., the TRC 1-2 recognitionsequence). In particular examples, the CAR transgene is inserted intoSEQ ID NO: 58 between nucleotide positions 13 and 14.

In some of those embodiments wherein the genetically-modified immunecell expresses a CAR or exogenous TCR, such cells have no detectablecell-surface expression of an endogenous T cell receptor (e.g., analpha/beta T cell receptor). Thus, the invention further provides apopulation of genetically-modified immune cells that express a shRNAmiRand have no detectable cell-surface expression of an endogenous T cellreceptor (e.g., an alpha/beta T cell receptor), and in some embodimentsalso express a CAR or exogenous TCR. For example, the population caninclude a plurality of genetically-modified immune cells of theinvention which express a CAR (i.e., are CAR+), or an exogenous T cellreceptor (i.e., exoTCR+), and have no cell-surface expression of anendogenous T cell receptor (i.e., are TCR−).

As used herein, “detectable cell-surface expression of an endogenousTCR” refers to the ability to detect one or more components of the TCRcomplex (e.g., an alpha/beta TCR complex) on the cell surface of animmune cell using standard experimental methods. Such methods caninclude, for example, immunostaining and/or flow cytometry specific forcomponents of the TCR itself, such as a TCR alpha or TCR beta chain, orfor components of the assembled cell-surface TCR complex, such as CD3.Methods for detecting cell-surface expression of an endogenous TCR(e.g., an alpha/beta TCR) on an immune cell include those described inthe examples herein, and, for example, those described in MacLeod et al.(2017) Molecular Therapy 25(4): 949-961.

2.4 shRNAmiR Target Proteins

The genetically-modified immune cells of the presently disclosedcompositions and methods can comprise and express a shRNAmiR thatreduces the expression of any endogenous protein. Non-limiting examplesof endogenous proteins whose expression can be reduced with a shRNAmiRinclude beta-2 microglobulin (B2M), transforming growth factor betareceptor 2 (TGFBR2), Cbl proto-oncogene B (CBL-B), CS1, CD52,deoxycytidine kinase (DCK), glucocorticoid receptor (GR), a T cellreceptor alpha gene, and a T cell receptor alpha constant region gene.

A. Beta-2 Microglobulin

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule is B2M.B2M is a component of the major histocompatibility complex (MHC) class Imolecule, which will not assemble on the cell surface without B2Mpresent. MHC class I molecules are comprised of α1, α2, and α3 proteins,in addition to B2M. Within MHC class I molecules, the B2M protein issituated beside the α3 protein and below the α1 protein on the cellsurface. B2M lacks a transmembrane region and is necessary for thestability of the peptide-binding groove of MHC class I molecules.

The shRNAmiR molecule may target any region of a B2M mRNA.Representative B2M mRNA and protein sequences are known in the art. Anon-limiting example of a B2M mRNA sequence is NCBI Acc. No. NM_004048.3and a B2M protein sequence is NCBI Acc. No. NP_004039.1.

In some of those embodiments wherein the expression of B2M is reduced bya shRNAmiR, the cell surface expression of B2M is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell (e.g., a cellnot expressing a B2M-targeted shRNAmiR).

Given that B2M is necessary for the assembly of the MHC class I moleculeon the cell surface, cells with reduced expression of B2M also exhibit areduction in MHC class I molecules on the cell surface. In some of theseembodiments, the expression of MHC class I molecules is reduced on thecell surface by at least about 10%, about 20%, about 30%, about 40%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or up to about 99% compared to acontrol cell (e.g., a cell not expressing a B2M-targeted shRNAmiR).

shRNAmiR molecules that target B2M may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the B2M gene. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 17 and 18, respectively (e.g., B2M 7289 shRNAmiR). Inparticular embodiments, the passenger and guide sequence of the shRNAmiRcomprise the sequences set forth as SEQ ID NO: 7 and 8, respectively(e.g., B2M 7282 shRNAmiR). In other embodiments, the passenger and guidesequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO:9 and 10, respectively (e.g., B2M 7285 shRNAmiR). In still otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 11 and 12, respectively (e.g., B2M7286 shRNAmiR). In yet other embodiments, the passenger and guidesequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO:13 and 14, respectively (e.g., B2M 7287 shRNAmiR). In particularembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 15 and 16, respectively (e.g., B2M7288 shRNAmiR). In certain embodiments, the passenger and guide sequenceof the shRNAmiR comprise the sequences set forth as SEQ ID NO: 19 and20, respectively (e.g., B2M 7290 shRNAmiR).

The B2M-targeted shRNAmiR may comprise a sequence having at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to the nucleic acidsequence set forth in any one of SEQ ID NOs: 41-47. In particularembodiments, the shRNAmiR comprises the sequence set forth in any one ofSEQ ID NOs: 41-47. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 41. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 42. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 43. In someembodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO:44. In some embodiments, the shRNAmiR comprises the sequence set forthin SEQ ID NO: 45. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 46. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 47.

Cells with reduced levels of B2M and MEW class I molecules can exhibitreduced allogenicity compared to a control cell (e.g., a cell notexpressing a B2M-targeted shRNAmiR). As used herein, the term“allogenicity” refers to the ability of a cell to be recognized andacted upon by the immune system as “other” or not autologous.Allogenicity can be measured using any method known in the art,including those methods described elsewhere herein wherein thepercentage of living cells were quantitated after incubation with primedalloantigen-specific CTLs or NK cells.

B. CS1

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule is CS1(also known as CCND3 subset 1, CRACC, CD319, and SLAMF7). CS1 is amember of the signaling lymphocyte activating-molecule (SLAM)-relatedreceptor family and is expressed on the surface of normal NK cells, Bcells, T cells, dendritic cells, NK-T cells, and monocytes. CS1 isoverexpressed by multiple myeloma cells and can serve as animmunotherapeutic target for multiple myeloma.

The shRNAmiR molecule may target any region of a CS1 mRNA.Representative CS1 mRNA and protein sequences are known in the art. Anon-limiting example of a CS1 mRNA sequence is NCBI Acc. No. NM_021181and a CS1 protein sequence is NCBI Acc. No. NP_067004.3.

In some of those embodiments wherein the expression of CS1 is reduced bya shRNAmiR, the cell surface expression of CS1 is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell (e.g., a cellnot expressing a CS1-targeted shRNAmiR).

shRNAmiR molecules that target CS1 may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the CS1 gene. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 21 and 22, respectively. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 23 and 24, respectively. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 25 and 26, respectively.

The CS1-targeted shRNAmiR may comprise a sequence having at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to the nucleic acidsequence set forth in any one of SEQ ID NOs: 48-50. In particularembodiments, the shRNAmiR comprises the sequence set forth in any one ofSEQ ID NOs: 48-50. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 48. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 49. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 50.

In some of the embodiments wherein the genetically-modified cellexpresses a shRNAmiR that reduces the expression of CS1, thegenetically-modified immune cell comprises a CAR having specificity forCS1. Non-limiting examples of CARs having specificity for CS1 include,without limitation, those described in WO 2014/179759, WO2015121454, andWO 2016/090369.

Cells having the expression of CS1 knocked down via shRNAmiR expressioncan be less susceptible to fratricide by a genetically-modified immunecell expressing a CAR having specificity for CS1 as compared to acontrol cell (e.g., a cell not expressing a CS1-targeted shRNAmiR). Thisis useful when using CAR-expressing cells with specificity for CS1 forthe treatment of a disease, such as multiple myeloma, wherein prolongedpresence of the CAR-expressing cell and thus, killing of the diseasedcells (e.g., multiple myeloma cell) is desired. As used herein, the term“fratricide” refers to the killing of cells by cells of like genotypeand/or phenotype. Fratricide by a genetically-modified immune cellexpressing a CS1-specific CAR can be measured using any method known inthe art, including but not limited to incubation of immune cellsexpressing the CS1-specific shRNAmiR with immune cells expressing aCS1-specific CAR and quantitating the number of livingshRNAmiR-expressing cells.

C. TGFBR2

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule istransforming growth factor beta receptor 2 (TGFBR2). TGFBR2 is atransmembrane receptor that binds transforming growth factor-beta(TGFB). TGFBR2 comprises a serine/threonine protein kinase domain andheterodimerizes with other TGFB receptors.

The shRNAmiR molecule may target any region of a TGFBR2 mRNA.Representative B2M mRNA and protein sequences are known in the art. Anon-limiting example of a TGFBR2 mRNA sequence is NM_001024847.2 and aTGFBR2 protein sequence is NCBI Acc. No. NP_001020018.1.

In some of those embodiments wherein the expression of TGFBR2 is reducedby a shRNAmiR, the cell surface expression of TGFBR2 is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or up to about 99% compared to a control cell (e.g., acell not expressing a TGFBR2-targeted shRNAmiR).

shRNAmiR molecules that target TGFBR2 may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the TGFBR2 gene. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 27 and 28, respectively (e.g., TGFBR2 721110 shRNAmiR). Inother embodiments, the passenger and guide sequence of the shRNAmiRcomprise the sequences set forth as SEQ ID NO: 29 and 30, respectively(e.g., TGFBR2 721111 shRNAmiR). In still other embodiments, thepassenger and guide sequence of the shRNAmiR comprise the sequences setforth as SEQ ID NO: 31 and 32, respectively (e.g., TGFBR2 721112shRNAmiR). In yet other embodiments, the passenger and guide sequence ofthe shRNAmiR comprise the sequences set forth as SEQ ID NO: 33 and 34,respectively (e.g., TGFBR2 721113 shRNAmiR). In particular embodiments,the passenger and guide sequence of the shRNAmiR comprise the sequencesset forth as SEQ ID NO: 35 and 36, respectively (e.g., TGFBR2 721114shRNAmiR).

The TGFBR2-targeted shRNAmiR may comprise a sequence having at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or more sequence identity to thenucleic acid sequence set forth in any one of SEQ ID NOs: 51-55. Inparticular embodiments, the shRNAmiR comprises the sequence set forth inany one of SEQ ID NOs: 51-55. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 51. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 52. In someembodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO:53. In some embodiments, the shRNAmiR comprises the sequence set forthin SEQ ID NO: 54. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 55.

In some of the embodiments wherein the genetically-modified immune cellexpresses a shRNAmiR that reduces the expression of TGFBR2, thegenetically-modified immune cell is less susceptible toimmunosuppression by transforming growth factor B1 (TGFB1) compared to acontrol cell (e.g., an immune cell not expressing a TGFBR2-targetedshRNAmiR). As used herein, the term “immunosuppression” refers to thereduction of the activation or efficacy of the immune system.Immunosuppression by TGFB1 can be measured using any method known in theart, including but not limited to measuring the effects of TGFB1 on Tcell differentiation and/or cytokine production.

D. CBL-B

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule is Cblproto-oncogene B (CBL-B). CBL-B is an E3 ubiquitin ligase that catalyzesthe attachment of ubiquitin to a protein, thus targeting the protein fordegradation.

The shRNAmiR molecule may target any region of a CBL-B mRNA.Representative CBL-B mRNA and protein sequences are known in the art. Anon-limiting example of a CBL-B mRNA sequence is NCBI Acc. No.NM_170662.5 and a CBL-B protein sequence is NCBI Acc. No. NP_733762.2.

In some of those embodiments wherein the expression of CBL-B is reducedby a shRNAmiR, the cell surface expression of CBL-B is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or up to about 99% compared to a control cell (e.g., acell not expressing a CBL-B-targeted shRNAmiR).

shRNAmiR molecules that target CBL-B may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the CBL-B gene.

In some of the embodiments wherein the genetically-modified immune cellexpresses a shRNAmiR that reduces the expression of CBL-B, thegenetically-modified immune cell is less susceptible to suppression of Tcell receptor (TCR) signaling by degradation of downstream signalingproteins compared to a control cell (e.g., an immune cell not expressinga CBL-B-targeted shRNAmiR). CBL proteins regulates the turnover of p85(a regulatory subunit of PI3K), phospholipase C-g, and tyrosine kinases,such as Lck, Fyn, and ZAP70, all of which are involved in TCR signaling(Lee et al. (2003) Science 302:1218-1222). Susceptibility to suppressionof TCR signaling by degradation of downstream signaling proteins can bemeasured using any method known in the art, including but not limited toELISA, flow cytometry, Western blot, immunocytochemistry, andimmunoprecipitation.

E. CD52

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule is CD52(cluster of differentiation 52), which is also known as CAmPATH-1antigen. CD52 is a glycoprotein present on the surface of maturelymphocytes and on monocytes and dendritic cells. Soluble CD52 moleculesinteract with sialic acid-binding immunoglobulin-like lectin 10(Siglec10) to inhibit T cell proliferation and activation (Zhao et al.(2017) Inflamm Res 66(7):571-578).

The shRNAmiR molecule may target any region of a CD52 mRNA.Representative CD52 mRNA and protein sequences are known in the art. Anon-limiting example of a CD52 mRNA sequence is NCBI Acc. No.NM_001803.3 and a CD52 protein sequence is NCBI Acc. No. NP_001794.2.

In some of those embodiments wherein the expression of CD52 is reducedby a shRNAmiR, the cell surface expression of CD52 is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or up to about 99% compared to a control cell (e.g., acell not expressing a CD52-targeted shRNAmiR).

shRNAmiR molecules that target CD52 may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the CD52 gene. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 37 and 38, respectively (e.g., CD52 72123 shRNAmiR). In otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 39 and 40, respectively (e.g.,CD52 72124 shRNAmiR).

The CD52-targeted shRNAmiR may comprise a sequence having at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to the nucleic acidsequence set forth as SEQ ID NO: 56 or 57. In particular embodiments,the shRNAmiR comprises the sequence set forth as SEQ ID NO: 56 or 57. Insome embodiments, the shRNAmiR comprises the sequence set forth in SEQID NO: 56. In some embodiments, the shRNAmiR comprises the sequence setforth in SEQ ID NO: 57.

In some of the embodiments wherein the genetically-modified immune cellexpresses a shRNAmiR that reduces the expression of CD52, thegenetically-modified immune cell is less susceptible to CD52antibody-induced cell death.

F. Deoxycytidine Kinase (DCK)

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule isdeoxycytidine kinase (DCK). DCK predominantly phosphorylatesdeoxycytidine and converts it into deoxycytidine monophosphate.

The shRNAmiR molecule may target any region of a DCK mRNA.Representative DCK mRNA and protein sequences are known in the art. Anon-limiting example of a DCK mRNA sequence is NCBI Acc. No. NM_000788.3and a DCK protein sequence is NCBI Acc. No. NP_000779.1.

In some of those embodiments wherein the expression of DCK is reduced bya shRNAmiR, the cell surface expression of DCK is reduced by at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or up to about 99% compared to a control cell (e.g., a cellnot expressing a DCK-targeted shRNAmiR).

shRNAmiR molecules that target DCK may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the DCK gene. In some embodiments, the passengerand guide sequence of the shRNAmiR comprise the sequences set forth asSEQ ID NO: 76 and 77, respectively (e.g., DCK 72136 shRNAmiR). In otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 78 and 79, respectively (e.g., DCK72137 shRNAmiR). In other embodiments, the passenger and guide sequenceof the shRNAmiR comprise the sequences set forth as SEQ ID NO: 80 and81, respectively (e.g., DCK 72138 shRNAmiR). In other embodiments, thepassenger and guide sequence of the shRNAmiR comprise the sequences setforth as SEQ ID NO: 82 and 83, respectively (e.g., DCK 72139 shRNAmiR).In other embodiments, the passenger and guide sequence of the shRNAmiRcomprise the sequences set forth as SEQ ID NO: 84 and 85, respectively(e.g., DCK 72140 shRNAmiR).

The DCK-targeted shRNAmiR may comprise a sequence having at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to the nucleic acidsequence set forth in any one of SEQ ID NOs: 86-90. In particularembodiments, the shRNAmiR comprises the sequence set forth in any one ofSEQ ID NOs: 86-90. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 86. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 87. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 88. In someembodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO:89. In some embodiments, the shRNAmiR comprises the sequence set forthin SEQ ID NO: 90.

In some of the embodiments wherein the genetically-modified immune cellexpresses a shRNAmiR that reduces the expression of DCK, thegenetically-modified immune cell is less susceptible to effects ofpurine nucleoside analogs (e.g., fludarabine) on cell proliferation.Indeed, genetically-modified immune cells having reduced expression ofDCK can be enriched by incubation of a cell population with a purinenucleoside analog such as fludarabine. Further, genetically-modifiedimmune cells (e.g., CAR T cells) having reduced expression of DCK mayhave greater persistence in vivo during immunotherapy when a purinenucleoside analog such as fludarabine is administered during the courseof therapy.

G. Glucocorticoid Receptor (GR)

In some embodiments, the endogenous protein with reduced expressionlevels as the result of the expression of a shRNAmiR molecule isglucocorticoid receptor (GR). Binding of glucocorticoids, such ascortisol or dexamethasone, can induce the release of protein, such asheat shock proteins, that can lead to transactivation or transrepressionin the cell.

The shRNAmiR molecule may target any region of a GR mRNA. RepresentativeGR mRNA and protein sequences are known in the art. A non-limitingexample of a GR mRNA sequence is NCBI Acc. No. AM183262.1.

In some of those embodiments wherein the expression of GR is reduced bya shRNAmiR, the expression of GR is reduced by at least about 10%, about20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or upto about 99% compared to a control cell (e.g., a cell not expressing aGR-targeted shRNAmiR).

shRNAmiR molecules that target GR may comprise any passenger andcorresponding guide sequence that is complementary (fully or partially)to a sequence within the GR gene. In some embodiments, the passenger andguide sequence of the shRNAmiR comprise the sequences set forth as SEQID NO: 91 and 92, respectively (e.g., GR 72142 shRNAmiR). In otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 93 and 94, respectively (e.g., GR72143 shRNAmiR). In other embodiments, the passenger and guide sequenceof the shRNAmiR comprise the sequences set forth as SEQ ID NO: 95 and96, respectively (e.g., GR 72145 shRNAmiR). In other embodiments, thepassenger and guide sequence of the shRNAmiR comprise the sequences setforth as SEQ ID NO: 97 and 98, respectively (e.g., GR 72146 shRNAmiR).In other embodiments, the passenger and guide sequence of the shRNAmiRcomprise the sequences set forth as SEQ ID NO: 99 and 100, respectively(e.g., GR 72148 shRNAmiR). In other embodiments, the passenger and guidesequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO:101 and 102, respectively (e.g., GR 72149 shRNAmiR). In otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 103 and 104, respectively (e.g.,GR 72150 shRNAmiR). In other embodiments, the passenger and guidesequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO:105 and 106, respectively (e.g., GR 72151 shRNAmiR). In otherembodiments, the passenger and guide sequence of the shRNAmiR comprisethe sequences set forth as SEQ ID NO: 107 and 108, respectively (e.g.,GR 72152 shRNAmiR).

The GR-targeted shRNAmiR may comprise a sequence having at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to the nucleic acidsequence set forth in any one of SEQ ID NOs: 109-117. In particularembodiments, the shRNAmiR comprises the sequence set forth in any one ofSEQ ID NOs: 109-117. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 109. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 110. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 111. In someembodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO:112. In some embodiments, the shRNAmiR comprises the sequence set forthin SEQ ID NO: 113. In some embodiments, the shRNAmiR comprises thesequence set forth in SEQ ID NO: 114. In some embodiments, the shRNAmiRcomprises the sequence set forth in SEQ ID NO: 115. In some embodiments,the shRNAmiR comprises the sequence set forth in SEQ ID NO: 116. In someembodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO:117.

In some of the embodiments wherein the genetically-modified immune cellexpresses a shRNAmiR that reduces the expression of GR, thegenetically-modified immune cell is less susceptible to effects ofglucocorticoids, such as cortisol or dexamethasone, such as reducedproliferation. Indeed, genetically-modified immune cells having reducedexpression of GR can be enriched by incubation of a cell population witha glucocorticoid. Further, genetically-modified immune cells (e.g., CARTcells) having reduced expression of GR may have greater persistence invivo during immunotherapy when a glucocorticoid (e.g., steroid) isadministered during the course of therapy.

2.5 Methods for Reducing Expression of Endogenous Proteins

The present invention provides methods for reducing the expression of anendogenous protein in an immune cell by introducing into the cell atemplate nucleic acid comprising a nucleic acid sequence encoding ashRNAmiR, whereby the template nucleic acid is inserted into the genomeand expressed.

The template nucleic acid can be inserted into the genome of the immunecell by random integration. Alternatively, the template nucleic acid canbe inserted into a target gene by nuclease-mediated targeted insertion,wherein an engineered nuclease has specificity for a recognitionsequence in the genome of the immune cell and generates a cleavage siteat the recognition sequence, allowing for the insertion of the templatenucleic acid into the genome of the immune cell at the cleavage site.

Any engineered nuclease can be used for targeted insertion of thetemplate nucleic acid, including an engineered meganuclease, a zincfinger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, ora megaTAL.

For example, zinc-finger nucleases (ZFNs) can be engineered to recognizeand cut pre-determined sites in a genome. ZFNs are chimeric proteinscomprising a zinc finger DNA-binding domain fused to a nuclease domainfrom an endonuclease or exonuclease (e.g., Type IIs restrictionendonuclease, such as the FokI restriction enzyme). The zinc fingerdomain can be a native sequence or can be redesigned through rational orexperimental means to produce a protein which binds to a pre-determinedDNA sequence ˜18 basepairs in length. By fusing this engineered proteindomain to the nuclease domain, it is possible to target DNA breaks withgenome-level specificity. ZFNs have been used extensively to target geneaddition, removal, and substitution in a wide range of eukaryoticorganisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978(2005)).

Likewise, TAL-effector nucleases (TALENs) can be generated to cleavespecific sites in genomic DNA. Like a ZFN, a TALEN comprises anengineered, site-specific DNA-binding domain fused to an endonuclease orexonuclease (e.g., Type IIs restriction endonuclease, such as the FokIrestriction enzyme) (reviewed in Mak, et al. (2013) Curr Opin StructBiol. 23:93-9). In this case, however, the DNA binding domain comprisesa tandem array of TAL-effector domains, each of which specificallyrecognizes a single DNA basepair.

Compact TALENs are an alternative endonuclease architecture that avoidsthe need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4:1762).A Compact TALEN comprises an engineered, site-specific TAL-effectorDNA-binding domain fused to the nuclease domain from the I-TevI homingendonuclease or any of the endonucleases listed in Table 2 in U.S.Application No. 20130117869. Compact TALENs do not require dimerizationfor DNA processing activity, so a Compact TALEN is functional as amonomer.

Engineered endonucleases based on the CRISPR/Cas system are also knownin the art (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al.(2013) Nat Methods. 10:957-63). A CRISPR system comprises twocomponents: (1) a CRISPR nuclease; and (2) a short “guide RNA”comprising a ˜20 nucleotide targeting sequence that directs the nucleaseto a location of interest in the genome. The CRISPR system may alsocomprise a tracrRNA. By expressing multiple guide RNAs in the same cell,each having a different targeting sequence, it is possible to target DNAbreaks simultaneously to multiple sites in the genome.

Engineered meganucleases that bind double-stranded DNA at a recognitionsequence that is greater than 12 base pairs can be used for thepresently disclosed methods. A meganuclease can be an endonuclease thatis derived from I-CreI and can refer to an engineered variant of I-CreIthat has been modified relative to natural I-CreI with respect to, forexample, DNA-binding specificity, DNA cleavage activity, DNA-bindingaffinity, or dimerization properties. Methods for producing suchmodified variants of I-CreI are known in the art (e.g. WO 2007/047859,incorporated by reference in its entirety). A meganuclease as usedherein binds to double-stranded DNA as a heterodimer. A meganuclease mayalso be a “single-chain meganuclease” in which a pair of DNA-bindingdomains is joined into a single polypeptide using a peptide linker.

Nucleases referred to as megaTALs are single-chain endonucleasescomprising a transcription activator-like effector (TALE) DNA bindingdomain with an engineered, sequence-specific homing endonuclease.

In particular embodiments, the recognition sequence of the engineerednuclease is within a target gene. The target gene can be any gene inwhich the sequence is desired to be altered (e.g., addition orsubtraction of nucleotide, substitution of nucleotide, or insertion of aheterologous or exogenous sequence). For example, knockout of a targetgene by genetic inactivation may be desired. In some embodiments, thetarget gene is a TCR alpha gene or a TCR beta gene. In particularembodiments, the target gene can be the TCR alpha constant region (TRAC)gene. In some specific embodiments, the target gene is the TRAC gene andthe recognition sequence is the TRC 1-2 recognition sequence set forthin SEQ ID NO: 58.

In some of these embodiments, the insertion of the template nucleic acidinto the target gene leads to disruption of expression of thefull-length endogenous protein encoded by the target gene. Thus, in someof those embodiments wherein the target gene is a TCR alpha gene, TRACgene, or TCR beta gene, the genetically-modified immune cell does nothave detectable cell-surface expression of an endogenous TCR, such as analpha/beta TCR, because the endogenous TCR will not properly assemble atthe cell surface in the absence of the endogenous proteins encoded bythese genes.

In particular embodiments in which the genetically-modified immune celldoes not have detectable cell-surface expression of an endogenous TCR(e.g., an alpha/beta TCR) due to inactivation of a gene encoding acomponent of an alpha/beta TCR, the genetically-modified immune cellfurther expresses a CAR or exogenous TCR and/or an HLA-E fusion protein.The CAR or exogenous TCR and/or the HLA-E fusion protein can be encodedby sequences comprised within the template nucleic acid. In some ofthese embodiments, the CAR/TCR-encoding sequence and/or the HLA-E fusionprotein-encoding sequence is operably linked to a different promoterthan the shRNAmiR-encoding sequence. In alternative embodiments, theCAR/TCR-encoding sequence and/or the HLA-E fusion protein-encodingsequence is operably linked to the same promoter, or to a differentpromoter, as the shRNAmiR-encoding sequence. The CAR/TCR-encodingsequence and/or the HLA-E fusion protein-encoding sequence can be 5′ or3′ of the shRNAmiR-encoding sequence, and the coding sequences can be inthe same or different orientation, such as 5′ to 3′ or 3′ to 5′.Further, the coding sequences may be separated by an element known inthe art to allow for the translation of two or more genes (i.e.,cistrons) from the same nucleic acid molecule including, but not limitedto, an IRES element, a T2 A element, a P2 A element (e.g., a P2A/furin), an E2 A element, and an F2 A element.

The use of nucleases for disrupting expression of an endogenous TCR hasbeen disclosed, including the use of zinc finger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs), megaTALs, andCRISPR systems (e.g., Osborn et al. (2016), Molecular Therapy 24(3):570-581; Eyquem et al. (2017), Nature 543: 113-117; U.S. Pat. No.8,956,828; U.S. Publication No. US2014/0301990; U.S. Publication No.US2012/0321667). The specific use of engineered meganucleases forcleaving DNA targets in the human TRAC gene has also been previouslydisclosed. For example, International Publication No. WO 2014/191527,which disclosed variants of the I-OnuI meganuclease that were engineeredto target a recognition sequence within exon 1 of the TCR alpha constantregion gene.

Moreover, in International Publication Nos. WO 2017/062439 and WO2017/062451, Applicants disclosed engineered meganucleases which havespecificity for recognition sequences in exon 1 of the TCR alphaconstant region (TRAC) gene. These included “TRC 1-2 meganucleases”which have specificity for the TRC 1-2 recognition sequence (SEQ ID NO:58) in exon 1 of the TRAC gene. The '439 and '451 publications alsodisclosed methods for targeted insertion of a CAR coding sequence or anexogenous TCR coding sequence into the TCR 1-2 meganuclease cleavagesite.

In particular embodiments, the nucleases used to practice the inventionare single-chain meganucleases. A single-chain meganuclease comprises anN-terminal subunit and a C-terminal subunit joined by a linker peptide.Each of the two domains recognizes half of the recognition sequence(i.e., a recognition half-site) and the site of DNA cleavage is at themiddle of the recognition sequence near the interface of the twosubunits. DNA strand breaks are offset by four base pairs such that DNAcleavage by a meganuclease generates a pair of four base pair, 3′single-strand overhangs.

Engineered nucleases can be delivered into a cell in the form of proteinor, preferably, as a nucleic acid encoding the engineered nuclease. Suchnucleic acids can be DNA (e.g., circular or linearized plasmid DNA orPCR products) or RNA (e.g., mRNA). For embodiments in which theengineered nuclease coding sequence is delivered in DNA form, it shouldbe operably linked to a promoter to facilitate transcription of thenuclease gene. Mammalian promoters suitable for the invention includeconstitutive promoters such as the cytomegalovirus early (CMV) promoter(Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or theSV40 early promoter (Benoist and Chambon (1981), Nature.290(5804):304-10) as well as inducible promoters such as thetetracycline-inducible promoter (Dingermann et al. (1992), Mol CellBiol. 12(9):4038-45). A nucleic acid encoding an engineered nuclease canalso be operably linked to a synthetic promoter. Synthetic promoters caninclude, without limitation, the JeT promoter (WO 2002/012514).

In certain embodiments, a nucleic acid sequence encoding an engineerednuclease is delivered on a recombinant DNA construct or expressioncassette. For example, the recombinant DNA construct can comprise anexpression cassette (i.e., “cassette”) comprising a promoter and anucleic acid sequence encoding an engineered nuclease described herein.

In some embodiments, mRNA encoding the engineered nuclease is deliveredto the cell because this reduces the likelihood that the gene encodingthe engineered nuclease will integrate into the genome of the cell.

The mRNA encoding an engineered nuclease can be produced using methodsknown in the art such as in vitro transcription. In some embodiments,the mRNA comprises a modified 5′ cap. Such modified 5′ caps are known inthe art and can include, without limitation, an anti-reverse cap analogs(ARCA) (U.S. Pat. No. 7,074,596), 7-methyl-guanosine, CLEANCAP reversecap analogs, such as Cap 1 analogs (Trilink; San Diego, Calif.), orenzymatically capped using, for example, a vaccinia capping enzyme orthe like. In some embodiments, the mRNA may be polyadenylated. The mRNAmay contain various 5′ and 3′ untranslated sequence elements to enhanceexpression of the encoded engineered nuclease and/or stability of themRNA itself. Such elements can include, for example, posttranslationalregulatory elements such as a woodchuck hepatitis virusposttranslational regulatory element.

The mRNA may contain nucleoside analogs or naturally-occurringnucleosides, such as pseudouridine, 5-methylcytidine,N6-methyladenosine, 5-methyluridine, or 2-thiouridine. Additionalnucleoside analogs include, for example, those described in U.S. Pat.No. 8,278,036.

In another particular embodiment, a nucleic acid encoding an engineerednuclease can be introduced into the cell using a single-stranded DNAtemplate. The single-stranded DNA can further comprise a 5′ and/or a 3′AAV inverted terminal repeat (ITR) upstream and/or downstream of thesequence encoding the engineered nuclease. In other embodiments, thesingle-stranded DNA can further comprise a 5′ and/or a 3′ homology armupstream and/or downstream of the sequence encoding the engineerednuclease.

In another particular embodiment, genes encoding a nuclease can beintroduced into a cell using a linearized DNA template. In someexamples, a plasmid DNA encoding a nuclease can be digested by one ormore restriction enzymes such that the circular plasmid DNA islinearized prior to being introduced into a cell.

Purified nuclease proteins can be delivered into cells to cleave genomicDNA, which allows for homologous recombination or non-homologousend-joining at the cleavage site with a sequence of interest, by avariety of different mechanisms known in the art, including thosefurther detailed herein below.

In some embodiments, nuclease proteins, or DNA/mRNA encoding thenuclease, are coupled to a cell penetrating peptide or targeting ligandto facilitate cellular uptake. Examples of cell penetrating peptidesknown in the art include poly-arginine (Jearawiriyapaisarn, et al.(2008) Mol Ther. 16:1624-9), TAT peptide from the HIV virus (Hudecz etal. (2005), Med. Res. Rev. 25: 679-736), MPG (Simeoni, et al. (2003)Nucleic Acids Res. 31:2717-2724), Pep-1 (Deshayes et al. (2004)Biochemistry 43: 7698-7706), and HSV-1 VP-22 (Deshayes et al. (2005)Cell Mol Life Sci. 62:1839-49). In an alternative embodiment, nucleaseproteins, or DNA/mRNA encoding nucleases, are coupled covalently ornon-covalently to an antibody that recognizes a specific cell-surfacereceptor expressed on target cells such that the nucleaseprotein/DNA/mRNA binds to and is internalized by the target cells.Alternatively, nuclease protein/DNA/mRNA can be coupled covalently ornon-covalently to the natural ligand (or a portion of the naturalligand) for such a cell-surface receptor. (McCall, et al. (2014) TissueBarriers. 2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol.14:1264-74; Kang, et al. (2014) Curr Pharm Biotechnol. 15(3):220-30;Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11):1491-508).

In some embodiments, nuclease proteins, or DNA/mRNA encoding nucleases,are coupled covalently or, preferably, non-covalently to a nanoparticleor encapsulated within such a nanoparticle using methods known in theart (Sharma, et al. (2014) Biomed Res Int. 2014). A nanoparticle is ananoscale delivery system whose length scale is <1 μm, preferably <100nm. Such nanoparticles may be designed using a core composed of metal,lipid, polymer, or biological macromolecule, and multiple copies of thenuclease proteins, mRNA, or DNA can be attached to or encapsulated withthe nanoparticle core. This increases the copy number of theprotein/mRNA/DNA that is delivered to each cell and, so, increases theintracellular expression of each nuclease to maximize the likelihoodthat the target recognition sequences will be cut. The surface of suchnanoparticles may be further modified with polymers or lipids (e.g.,chitosan, cationic polymers, or cationic lipids) to form a core-shellnanoparticle whose surface confers additional functionalities to enhancecellular delivery and uptake of the payload (Jian et al. (2012)Biomaterials. 33(30): 7621-30). Nanoparticles may additionally beadvantageously coupled to targeting molecules to direct the nanoparticleto the appropriate cell type and/or increase the likelihood of cellularuptake. Examples of such targeting molecules include antibodies specificfor cell-surface receptors and the natural ligands (or portions of thenatural ligands) for cell surface receptors.

In some embodiments, the nuclease proteins or DNA/mRNA encoding thenucleases are encapsulated within liposomes or complexed using cationiclipids (see, e.g., Lipofectamine™, Life Technologies Corp., Carlsbad,Calif.; Zuris et al. (2015) Nat Biotechnol. 33: 73-80; Mishra et al.(2011) J Drug Deliv. 2011:863734). The liposome and lipoplexformulations can protect the payload from degradation, and facilitatecellular uptake and delivery efficiency through fusion with and/ordisruption of the cellular membranes of the target cells.

In some embodiments, nuclease proteins, or DNA/mRNA encoding nucleases,are encapsulated within polymeric scaffolds (e.g., PLGA) or complexedusing cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) TherDeliv. 2(4): 523-536). Polymeric carriers can be designed to providetunable drug release rates through control of polymer erosion and drugdiffusion, and high drug encapsulation efficiencies can offer protectionof the therapeutic payload until intracellular delivery to the desiredtarget cell population.

In some embodiments, nuclease proteins, or DNA/mRNA encoding recombinantnucleases, are combined with amphiphilic molecules that self-assembleinto micelles (Tong et al. (2007) J Gene Med. 9(11): 956-66). Polymericmicelles may include a micellar shell formed with a hydrophilic polymer(e.g., polyethyleneglycol) that can prevent aggregation, mask chargeinteractions, and reduce nonspecific interactions.

In some embodiments, nuclease proteins, or DNA/mRNA encodingmeganucleases, are formulated into an emulsion or a nanoemulsion (i.e.,having an average particle diameter of <1 nm) for administration and/ordelivery to the target cell. The term “emulsion” refers to, withoutlimitation, any oil-in-water, water-in-oil, water-in-oil-in-water, oroil-in-water-in-oil dispersions or droplets, including lipid structuresthat can form as a result of hydrophobic forces that drive apolarresidues (e.g., long hydrocarbon chains) away from water and polar headgroups toward water, when a water immiscible phase is mixed with anaqueous phase. These other lipid structures include, but are not limitedto, unilamellar, paucilamellar, and multilamellar lipid vesicles,micelles, and lamellar phases. Emulsions are composed of an aqueousphase and a lipophilic phase (typically containing an oil and an organicsolvent). Emulsions also frequently contain one or more surfactants.Nanoemulsion formulations are well known, e.g., as described in USPatent Application Nos. 2002/0045667 and 2004/0043041, and U.S. Pat.Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which isincorporated herein by reference in its entirety.

In some embodiments, nuclease proteins, or DNA/mRNA encoding nucleases,are covalently attached to, or non-covalently associated with,multifunctional polymer conjugates, DNA dendrimers, and polymericdendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845-56; Cheng etal. (2008) J Pharm Sci. 97(1): 123-43). The dendrimer generation cancontrol the payload capacity and size, and can provide a high drugpayload capacity. Moreover, display of multiple surface groups can beleveraged to improve stability, reduce nonspecific interactions, andenhance cell-specific targeting and drug release.

In some embodiments, genes encoding a nuclease are delivered using aviral vector (i.e., a recombinant virus). Such vectors are known in theart and include retroviral vectors, lentiviral vectors, adenoviralvectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci,et al. (2013 New Microbiol. 36:1-22). Recombinant AAV vectors useful inthe invention can have any serotype that allows for transduction of thevirus into the cell and insertion of the nuclease gene into the cellgenome. In particular embodiments, recombinant AAV vectors have aserotype of AAV2 or AAV6. AAV vectors can also be self-complementarysuch that they do not require second-strand DNA synthesis in the hostcell (McCarty, et al. (2001) Gene Ther. 8:1248-54). Polynucleotidesdelivered by recombinant AAV vectors, including those that deliver atemplate nucleic acid disclosed herein, can include left (5′) and right(3′) inverted terminal repeats.

If the nuclease genes are delivered in DNA form (e.g. plasmid) and/orvia a viral vector (e.g. AAV) they must be operably linked to apromoter. In some embodiments, this can be a viral promoter such asendogenous promoters from the viral vector (e.g. the LTR of a lentiviralvector) or the well-known cytomegalovirus- or SV40 virus-earlypromoters. In a preferred embodiment, nuclease genes are operably linkedto a promoter that drives gene expression preferentially in the targetcell (e.g., a T cell).

The CAR/TCR coding sequence and/or the HLA-E fusion protein codingsequence can further comprise additional control sequences. For example,the sequence can include homologous recombination enhancer sequences,Kozak sequences, polyadenylation sequences, transcriptional terminationsequences, selectable marker sequences (e.g., antibiotic resistancegenes), origins of replication, and the like. Sequences encodingengineered nucleases can also include at least one nuclear localizationsignal. Examples of nuclear localization signals are known in the art(see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105).

The invention further provides for the introduction of a templatenucleic acid into a target gene. In some embodiments, the templatenucleic acid comprises a 5′ homology arm and a 3′ homology arm flankingthe elements of the insert. Such homology arms have sequence homology tocorresponding sequences 5′ upstream and 3′ downstream of the nucleaserecognition sequence where a cleavage site is produced. In general,homology arms can have a length of at least 50 base pairs, preferably atleast 100 base pairs, and up to 2000 base pairs or more, and can have atleast 90%, preferably at least 95%, or more, sequence homology to theircorresponding sequences in the genome.

A template nucleic acid disclosed herein (e.g., encoding a shRNAmiR, anucleic acid encoding a CAR or exogenous TCR, and/or an HLA-E fusionprotein), can be introduced into the cell by any of the means previouslydiscussed. In a particular embodiment, the template nucleic acid isintroduced by way of a viral vector (i.e., a recombinant virus), such asa recombinant lentivirus, a recombinant retrovirus, a recombinantadenovirus, or preferably a recombinant AAV vector (i.e., a recombinantAAV). Recombinant AAV vectors useful for introducing an exogenousnucleic acid (e.g., a template nucleic acid) can have any serotype thatallows for transduction of the virus into the cell and insertion of theexogenous nucleic acid sequence into the cell genome. In particularembodiments, the recombinant AAV vectors have a serotype of AAV2 orAAV6. The recombinant AAV vectors can also be self-complementary suchthat they do not require second-strand DNA synthesis in the host cell.

In another particular embodiment, the template nucleic acid disclosedherein (e.g., encoding a shRNAmiR, a nucleic acid encoding a CAR orexogenous TCR, and/or an HLA-E fusion protein), can be introduced intothe cell using a single-stranded DNA template. The single-stranded DNAcan comprise the exogenous sequence of interest and, in preferredembodiments, can comprise 5′ and 3′ homology arms to promote insertionof the nucleic acid sequence into the meganuclease cleavage site byhomologous recombination. The single-stranded DNA can further comprise a5′ AAV inverted terminal repeat (ITR) sequence 5′ upstream of the 5′homology arm, and a 3′ AAV ITR sequence 3′ downstream of the 3′ homologyarm.

In another particular embodiment, the template nucleic acid disclosedherein (e.g., encoding a shRNAmiR, a nucleic acid encoding a CAR orexogenous TCR, and/or an HLA-E fusion protein) can be introduced intothe cell by transfection with a linearized DNA template. In someexamples, a plasmid DNA can be digested by one or more restrictionenzymes such that the circular plasmid DNA is linearized prior totransfection into the cell.

Immune cells (e.g., T cells) modified by the present invention mayrequire activation prior to introduction of a nuclease and/or anexogenous sequence of interest. For example, T cells can be contactedwith anti-CD3 and anti-CD28 antibodies that are soluble or conjugated toa support (i.e., beads) for a period of time sufficient to activate thecells.

Genetically-modified immune cells of the invention can be furthermodified to express one or more inducible suicide genes, the inductionof which provokes cell death and allows for selective destruction of thecells in vitro or in vivo. In some examples, a suicide gene can encode acytotoxic polypeptide, a polypeptide that has the ability to convert anon-toxic pro-drug into a cytotoxic drug, and/or a polypeptide thatactivates a cytotoxic gene pathway within the cell. That is, a suicidegene is a nucleic acid that encodes a product that causes cell death byitself or in the presence of other compounds. A representative exampleof such a suicide gene is one that encodes thymidine kinase of herpessimplex virus. Additional examples are genes that encode thymidinekinase of varicella zoster virus and the bacterial gene cytosinedeaminase that can convert 5-fluorocytosine to the highly toxic compound5-fluorouracil. Suicide genes also include as non-limiting examplesgenes that encode caspase-9, caspase-8, or cytosine deaminase. In someexamples, caspase-9 can be activated using a specific chemical inducerof dimerization (CID). A suicide gene can also encode a polypeptide thatis expressed at the surface of the cell that makes the cells sensitiveto therapeutic and/or cytotoxic monoclonal antibodies. In furtherexamples, a suicide gene can encode recombinant antigenic polypeptidecomprising an antigenic motif recognized by the anti-CD20 mAb Rituximaband an epitope that allows for selection of cells expressing the suicidegene. See, for example, the RQR8 polypeptide described in WO2013153391,which comprises two Rituximab-binding epitopes and a QBEnd10-bindingepitope. For such a gene, Rituximab can be administered to a subject toinduce cell depletion when needed. In further examples, a suicide genemay include a QBEnd10-binding epitope expressed in combination with atruncated EGFR polypeptide.

Variants of naturally-occurring nucleases and microRNA sequences(including pre-miRNA and pri-miRNA sequences) can be used in thepresently disclosed compositions and methods. As used herein, “variants”is intended to mean substantially similar sequences. A “variant”polypeptide is intended to mean a polypeptide derived from the “native”polypeptide by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native polypeptide. As usedherein, a “native” polynucleotide or polypeptide comprises a parentalsequence from which variants are derived. Variant polypeptidesencompassed by the embodiments are biologically active. That is, theycontinue to possess the desired biological activity of the nativeprotein. Such variants may result, for example, from human manipulation.Biologically active variants of a native polypeptide will have at leastabout 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99%, sequence identity to the amino acid sequence of the nativepolypeptide, as determined by sequence alignment programs and parametersdescribed elsewhere herein. A biologically active variant of apolypeptide may differ from that polypeptide or subunit by as few asabout 1-40 amino acid residues, as few as about 1-20, as few as about1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The polypeptides may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatdo not affect biological activity of the protein of interest may befound in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

For polynucleotides, a “variant” comprises a deletion and/or addition ofone or more nucleotides at one or more sites within the nativepolynucleotide. One of skill in the art will recognize that variants ofthe nucleic acids of the embodiments will be constructed such that theopen reading frame is maintained. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the embodiments. Variant polynucleotides include syntheticallyderived polynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a polypeptide or RNA.Generally, variants of a particular polynucleotide of the embodimentswill have at least about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein. Variants of a particularpolynucleotide (i.e., the reference polynucleotide) can also beevaluated by comparison of the percent sequence identity between thepolypeptide encoded by a variant polynucleotide and the polypeptideencoded by the reference polynucleotide.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by screening the polypeptide for its biologicalactivity.

2.6 Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a genetically-modified immune cell of the invention, or apopulation of genetically-modified immune cells of the invention, and apharmaceutically-acceptable carrier. Such pharmaceutical compositionscan be prepared in accordance with known techniques. See, e.g.,Remington, The Science and Practice of Pharmacy (21st ed. 2005). In themanufacture of a pharmaceutical formulation according to the invention,cells are typically admixed with a pharmaceutically acceptable carrierand the resulting composition is administered to a subject. The carriermust, of course, be acceptable in the sense of being compatible with anyother ingredients in the formulation and must not be deleterious to thesubject. In some embodiments, pharmaceutical compositions of theinvention can further comprise one or more additional agents useful inthe treatment of a disease in the subject. In additional embodiments,pharmaceutical compositions of the invention can further includebiological molecules, such as cytokines (e.g., IL-2, IL-7, IL-15, and/orIL-21), which promote in vivo cell proliferation and engraftment ofgenetically-modified T cells. Pharmaceutical compositions comprisinggenetically-modified immune cells of the invention can be administeredin the same composition as an additional agent or biological moleculeor, alternatively, can be co-administered in separate compositions.

The present disclosure also provides genetically-modified immune cells,or populations thereof, described herein for use as a medicament. Thepresent disclosure further provides the use of genetically-modifiedimmune cells or populations thereof described herein in the manufactureof a medicament for treating a disease in a subject in need thereof. Inone such aspect, the medicament is useful for cancer immunotherapy insubjects in need thereof.

Pharmaceutical compositions of the invention can be useful for treatingany disease state that can be targeted by adoptive immunotherapy, andparticularly T cell adoptive immunotherapy. In a particular embodiment,the pharmaceutical compositions and medicaments of the invention areuseful in the treatment of cancer. Non-limiting examples of cancerswhich may be treated with the pharmaceutical compositions andmedicaments of the present disclosure include, without limitation,various types of cancers described herein that can be targeted by a CARor exogenous TCR.

In some of these embodiments wherein cancer is treated with thepresently disclosed genetically-modified immune cells or populationsthereof, the subject administered the genetically-modified immune cellsor populations thereof is further administered an additionaltherapeutic, such as radiation, surgery, or a chemotherapeutic agent.

The invention further provides a population of genetically-modifiedimmune cells comprising a plurality of genetically-modified immune cellsdescribed herein, which comprise in their genome a nucleic acid sequenceencoding a shRNAmiR, wherein the exogenous nucleic acid moleculeencoding the shRNAmiR can be inserted into a target gene, such as theTCR alpha gene or the TRAC gene, such that the cell has no detectablecell-surface expression of an endogenous TCR (e.g., an alpha/beta TCR).Thus, in various embodiments of the invention, a population of immunecells is provided wherein at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or up to 100%, ofcells in the population are a genetically-modified immune cell describedherein. In some embodiments of the invention, a population of immunecells is provided wherein about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100%, ofcells in the population are a genetically-modified immune cell describedherein. In further embodiments of the invention, a population of immunecells is provided wherein at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or up to 100%, ofcells in the population are a genetically-modified immune cell describedherein which further expresses a CAR or exogenous TCR and/or furtherexpresses an HLA-E fusion protein. In certain embodiments of theinvention, a population of immune cells is provided wherein about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, or up to 100%, of cells in the population are agenetically-modified immune cell described herein which furtherexpresses a CAR or exogenous TCR and/or an HLA-E fusion protein.

2.7 Methods of Administering Genetically-Modified Immune Cells

Another aspect disclosed herein is the administration of an effectiveamount of the genetically-modified immune cells, or populations thereof,of the present disclosure to a subject in need thereof. In particularembodiments, the pharmaceutical compositions described herein areadministered to a subject in need thereof. For example, an effectiveamount of a population of cells can be administered to a subject havinga disease. In particular embodiments, the disease can be cancer, andadministration of the genetically-modified immune cells of the inventionrepresent an immunotherapy. The administered cells are able to reducethe proliferation, reduce the number, or kill target cells in therecipient. Unlike antibody therapies, genetically-modified immune cellsof the present disclosure are able to replicate and expand in vivo,resulting in long-term persistence that can lead to sustained control ofa disease.

Examples of possible routes of administration include parenteral, (e.g.,intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), orinfusion) administration. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. In specificembodiments, the agent is infused over a period of less than about 12hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In still otherembodiments, the infusion occurs slowly at first and then is increasedover time.

In some embodiments, a genetically-modified immune cell or populationthereof of the present disclosure targets a tumor antigen for thepurposes of treating cancer. Such cancers can include, withoutlimitation, various types of cancers described herein that can betargeted by a CAR or exogenous TCR.

When an “effective amount” or “therapeutic amount” is indicated, theprecise amount to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size (ifpresent), extent of infection or metastasis, and condition of thepatient (subject). In some embodiments, a pharmaceutical compositioncomprising the genetically-modified immune cells or populations thereofdescribed herein is administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, including all integer values within those ranges. In furtherembodiments, the dosage is 10⁵ to 10⁶ cells/kg body weight, includingall integer values within those ranges. In some embodiments, cellcompositions are administered multiple times at these dosages. The cellscan be administered by using infusion techniques that are commonly knownin immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In some embodiments, administration of genetically-modified immune cellsor populations thereof of the present disclosure reduce at least onesymptom of a target disease or condition. For example, administration ofgenetically-modified T cells or populations thereof of the presentdisclosure can reduce at least one symptom of a cancer. Symptoms ofcancers are well known in the art and can be determined by knowntechniques.

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Transient Knockdown of Beta-2 Microglobulin when shRNACassette is Inserted Into the Genome of Anti-CD19 CAR T Cells

In these experiments, it was assessed whether beta-2 microglobulin (B2M)can be efficiently knocked down using a single copy of an shRNA that isco-delivered to the T cell receptor alpha constant (TRAC) locus with theCAR gene. An apheresis sample was drawn from a healthy donor, and the Tcells were enriched using the CD3 positive selection kit II inaccordance with the manufacturer's instructions (Stem CellTechnologies). T cells were activated using ImmunoCult™ T cellstimulator (anti-CD2/CD3/CD28, Stem Cell Technologies) in X-VIVO™ 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1×10⁶ cells were electroporated with 1 μg of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence (SEQ ID NO: 58) in the TRAC gene, and weretransduced with AAV packaged with construct 7056 at an MOI of 25,000viral genomes/cell. AAV 7056 encodes a CAR (composed of the anti-CD19FMC63 scFv, CD8 hinge and transmembrane domains, a Novel 6 (N6)co-stimulatory domain, and a CD3 zeta intracellular signaling domain)oriented opposite of the TRAC open reading frame. Transcription isinitiated by the JeT promoter and terminated by a bi-directional poly-Asequence. Upstream of the JeT promoter controlling CAR expression issituated an shRNA expression cassette also in opposite transcriptionalorientation relative to the TRAC ORF. Transcription of the shRNA isinitiated by a U6 promoter and terminated by a central poly-pyrimidinetract. Several shRNA sequences were evaluated for knockdown potency ofB2M expression. AAV 7056 contains shRNA sequence TRCN0000381472,abbreviated as shRNA472, the sequence of which is set forth as SEQ IDNO: 6.

Cell cultures were maintained for up to 10 additional days in X-VIVO™ 15medium supplemented with 5% FBS and 30 ng/ml of IL-2. On days 4, 7,and/or 10 post-nucleofection, the cultures were sampled and analyzed forsurface expression of CD3 (anti-CD3-PE, BioLegend), CAR (anti-FMC63anti-CAR, clone VM16 conjugated to AlexaFluor488), B2M (anti-B2M-APC, orPE, clone TU-99 BD Biosciences), and HLA-A, B, and C (clone W6/32,BV605, BioLegend). Flow cytometry data were acquired on aBeckman-Coulter CytoFLEX-LX. The detection of cell-surface CD3 is anindicator of the endogenous T cell receptor on the cell surface.Accordingly, it is understood that genetically-modified cells which areCD3+ or CD3− are TCR+ and TCR−, respectively.

B2M and HLA-ABC levels were measured in samples expressing construct7056 and control populations. FIG. 1A shows the B2M surface levels inCD3−/CAR+ cells compared to TRAC-edited cells expressing no shRNA from acontrol culture. FIG. 1B shows B2M levels on CD3−/CAR+ versus CD3+/CAR−populations in the same culture. FIGS. 1C and 1D make the samerespective comparisons in displays of HLA-ABC surface levels. TheCD3−/CAR+ fraction of cells transduced with AAV-7056 displayed levels ofB2M and HLA-ABC that are reduced by greater than 90% compared to controlpopulations.

At the earliest time points following transgene delivery, CAR+ cellsdisplay a 90-95% reduction in surface levels of HLA-ABC and B2M,dependent on the marker and the comparison. As the culture is carriedout for longer periods of time, there is a reduction in the frequency ofCAR+ events and there is a recovery of B2M surface expression to nearnormal levels (FIGS. 2A-2F).

To determine the root cause of this loss of knockdown efficacy, thetransgenic insert in the genomes of surviving CAR+ cells were sequencedten days post-transduction (d10). Sequencing reactions were primed usingoligonucleotides that hybridize in the TRAC homology arms in the 7056sequence. The sequence of the self-complimentary hairpin structureintended to target the B2M transcript was not recovered in d10 genomes.However, this sequence was recovered from reactions in which the AAVpreparation or the packaging plasmid was used as templates. Sequenceloss appeared to be restricted to the hairpin sequence, as adjacentsequences, including the U6 promoter and the cPPT were not perturbed.

These studies show that a pre-screened B2M-targeted shRNA can knock downB2M expression levels on the surface of cells into which the constructhas been delivered (via targeted insertion into the T cell receptoralpha constant region locus). This effect is specific to CAR+populations (i.e., cells in which targeted integration into the TRAClocus has occurred). This experiment demonstrates that B2M can beefficiently knocked down using a single copy of shRNA472 co-delivered tothe TRAC locus with the CAR gene on the same AAV template. Althoughresults appeared promising at early time points, the knockdown effectwas determined to be transient and associated with toxicity or growtharrest in the CAR+ population. As the knockdown effect waned, a loss ofthe shRNA sequence from the genome of surviving T cells was observed.This suggested that shRNA472 was excised from genomes and is notsuitable for single integrated copy-mediated knockdown of B2M orpotentially other endogenous proteins.

Example 2 Design, Construction, and Characterization of Beta-2Microglobulin shRNAmiR

Targeting B2M expression using shRNA was hindered by transient knockdowneffects and toxicity in the CAR T product, owing perhaps to the loss ofthe hairpin sequence from the genome. The guide and passenger strandsequences comprising the short hairpin were adapted into a larger, morehighly structured micro-RNA scaffold (miR). This amalgam of shRNA andmiRNA technology is referred to herein as a microRNA-adapted shRNA orshRNAmiR.

The miR scaffold selected to generate the shRNAmiR is called miR-E,which is an engineered derivative of a naturally-occurring miR in thehuman genome called miR-30 (see International Publication No. WO2014/117050, which is herein incorporated by reference in its entirety).Micro-RNAs enter the cell's RNAi pathway in the nucleus, where they areprocessed by Drosha and exported into the cytosol by exportin complexesbefore interacting with Dicer and Argonaut, and loading into theRNA-induced silencing complex (RISC).

The sequences of the miR-E scaffold used in this study are set forth asSEQ ID NO: 1 (5′ miR-E scaffold domain), SEQ ID NO: 2 (5′ miR-E basalstem domain), SEQ ID NO: 3 (mir-30a loop domain), SEQ ID NO: 4 (3′ miR-Ebasal stem domain, and SEQ ID NO: 5 (3′ miR-E scaffold domain).B2M-targeting passenger and guide strand sequences, which are set forthas SEQ ID NOs: 7 and 8, respectively, were cloned into an anti-CD19 CAR(same as described in Example 1) vector downstream of the stop codon,but upstream of the poly-A transcriptional terminator. This vector(7282) was packaged into AAV6 capsids and used in a study to determinethe magnitude and duration of B2M knockdown.

In this study, an apheresis sample was drawn from a healthy donor, andthe T cells were enriched using the CD3 positive selection kit II inaccordance with the manufacturer's instructions (Stem CellTechnologies). T cells were activated using ImmunoCult™ T cellstimulator (anti-CD2/CD3/CD28, Stem Cell Technologies) in X-VIVO™ 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1×10⁶ cells were electroporated with 1 μg of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence in the TRAC gene, and were transduced with AAVpackaged with construct 7282 at an MOI of 25,000 viral genomes/cell. CART cells with no RNAi feature (7206) or with shRNA-472 (7056) wereincluded as controls.

At 3, 7, and 11 days after editing and AAV transduction, cells fromthese cultures were analyzed for TCR knockout (using anti-CD3-BB515clone SK7, BD Biosciences), CAR transgene knock-in (usinganti-FMC63-AlexaFluor647, clone VM16, produced in-house), and B2Mknockdown, using anti-B2M-PE (clone TU-99, BD Biosciences). B2M levels(measured by mean fluorescence intensity in the PE channel) werecompared between CD3−/CAR+ populations and CD3+/CAR− populations ofcells from the same culture.

At 3 days post-editing/transduction, CAR T cells produced using AAV 7282compared favorably with AAV 7206 in terms of CAR+ frequency, whilecultures produced using AAV 7056 contain a smaller frequency of CAR+cells. The frequency of CD3−/CAR+ cells increased or stayed the same incultures produced with AAV 7206 or AAV 7282, but were reduced to lessthan 5% of total cells in cultures transduced with AAV 7056.

CAR T cells expressing an shRNA (AAV 7056) rapidly downregulated surfacelevels of B2M by 91.9% on day 3. The B2M shRNAmiR downregulated B2Msurface levels by 77.4% at this time point (FIG. 3). As the cultureswere carried for additional days, the magnitude of knockdown mediated byshRNA decreased to 84.3% and 65.1% at days 7 and 11, respectively (FIGS.4 and 5). Furthermore, at these time points, there were CAR+ events inthe AAV 7056-transduced cultures that began to re-express normal or nearnormal levels of B2M. By comparison, the magnitude of knockdown mediatedby shRNAmiR surprisingly increased on days 7 and 11 to approximately 85%at both time points, with no upregulations in B2M expression observed.

Thus, the use of a shRNAmiR to interfere with B2M expressionadvantageously resulted in slower knockdown kinetics and a slightlylower knockdown magnitude, but a more stable phenotype and greatlyreduced toxicity when compared to the previously evaluated shRNAdescribed in Example 1. The superior results observed in this studyprovide an initial proof-of-concept for the use of shRNAmiRs todownregulate endogenous protein expression in CAR T cells, which may beadvantageous over gene knockout in certain situations.

Example 3 Design and Characterization of Additional B2M shRNAmiRConstructs

Six additional B2M guide and passenger strand sequences were identifiedand cloned into the miR-E backbone and inserted into the anti-CD19 CARconstruct described in Example 2 between the stop codon of the CAR andthe poly-A transcriptional terminator. The passenger and guide strandsof the B2M 7285 shRNAmiR are set forth as SEQ ID NOs: 9 and 10,respectively. The passenger and guide strands of the B2M 7286 shRNAmiRare set forth as SEQ ID NOs: 11 and 12, respectively. The passenger andguide strands of the B2M 7287 shRNAmiR are set forth as SEQ ID NOs: 13and 14, respectively. The passenger and guide strands of the B2M 7288shRNAmiR are set forth as SEQ ID NOs: 15 and 16, respectively. Thepassenger and guide strands of the B2M 7289 shRNAmiR are set forth asSEQ ID NOs: 17 and 18, respectively. The passenger and guide strands ofthe B2M 7290 shRNAmiR are set forth as SEQ ID NOs: 19 and 20,respectively. These additional B2M shRNAmiR sequences were tested fortheir ability to knockdown B2M expression.

In this study, an apheresis sample was drawn from a donor, and the Tcells were enriched using the CD3 positive selection kit II inaccordance with the manufacturer's instructions (Stem CellTechnologies). T cells were activated using ImmunoCult™ T cellstimulator (anti-CD2/CD3/CD28, Stem Cell Technologies) in X-VIVO™ 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1×10⁶ cells were electroporated with 1 μg of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence in the T cell receptor alpha constant locus. Thesix CAR-shRNAmiR constructs (constructs 7285-7290) were delivered to Tcells as linearized DNA (2 μg/sample), simultaneously with the TRC1-2RNA during nucleofection. Alternatively, T cells were nucleofected withthe B2M 13-14x.479 nuclease, which recognizes and cleaves the B2M 13-14recognition sequence (SEQ ID NO: 60) in the human B2M gene (see, WO2017112859). This was included to contextualize the amount of B2Msurface signal present in the shRNAmiR knockdowns by defining thebackground signal present on a B2M knockout cell.

At days 4 and 11 following nucleofection, samples of the cultures werestained with anti-CD3-BB515 (clone SK7, BD Biosciences),anti-FMC63-AlexaFluor647 (clone VM16, produced in-house), anti-B2M-PE(using clone TU-99, BD Biosciences). B2M levels (measured by meanfluorescence intensity in the PE channel) were compared betweenCD3−/CAR+ populations and CD3+/CAR− populations of cells from the sameculture.

The mean fluorescence intensity (MFI) of the CD3−/CAR+ population, aswell as the CD3+ CAR-population is listed beside the correspondingshRNAmiR in Table 1 below. Percent knockdown is defined as: (MFI of theshRNAmiR+ population/MFI of reference population)×100. Tabulated datawere acquired at day 11 post-nucleofection. Constructs 7289 and 7290exhibited the highest magnitude of B2M interference of the 7 constructstested. The background signal present on B2M knockout cells is less than2% of the reference population (not listed).

TABLE 1 Knockdown of B2M by candidate sequences. B2M MFI B2M MFI ControlConstruct CD3−/CAR+ population % knockdown 7002 208469 219415 5 728241478 218647 81 7285 125840 199812 37 7286 76390 228482 67 7287 90590222648 60 7288 31937 222326 86 7289 23676 228838 89 7290 23190 213868 89

All seven constructs tested in this study exhibited some degree ofstable B2M knockdown and CAR expression. Interestingly, this studydemonstrated that the degree of endogenous protein knockdown can bemodulated by the selection of different guide and passenger strands.This flexibility provided by the shRNAmiR approach can be advantageouswhen various degrees of endogenous protein knockdown may be preferableto a nearly complete knockout. Candidate sequences encoded in 7282,7288, 7289, and 7290 were investigated in further experiments.

Example 4 Allogenicity of shRNAmiR B2M CAR T Cells and Susceptibility toNatural Killer (NK) Cell Killing

These studies assessed the effects of a knockout of B2M via geneticablation in comparison to an incomplete, but stable knockdown usingshRNAmiR on the sensitivity of the cells to cytolysis byalloantigen-specific cytotoxic lymphocytes (CTLs) or NK cells. CTLs wereprimed using monocyte-derived dendritic cells from an unrelated healthydonor to activate and expand alloantigen-specific CD8+ T cellpopulations.

Apheresis samples were drawn from two unrelated healthy donors, and theT cells were enriched using the CD3 positive selection kit II inaccordance with the manufacturer's instructions (Stem CellTechnologies). To prepare dendritic cells, unfractionated mononuclearcells from the apheresis samples were plated in polystyrene cell cultureflasks and allowed to adhere for 1-2 hours. Nonadherent cells werediscarded and adherent cells were cultured for 6 days in a cytokinemixture that differentiates monocytes into dendritic cell-like cells(800 U/ml GM-CSF and 500 U/ml IL-4, both sourced from PeproTech).Dendritic cells (DCs) were collected and plated at various ratios with Tcells from unrelated donors. The first 24 h of co-culture were carriedout in the absence of exogenous cytokines. IL-2 was added to thecultures thereafter at 10 ng/ml.

Co-cultures were carried out for 5 days, and the CD8+ T cells wereenriched by depleting CD4+ T cells using Miltenyi CliniMACS CD4microbeads and an LS column. Purity was assessed using CD3-PE (CloneUCHT1, BioLegend) and CD8-BV421 (Clone RPA-T8, BioLegend). Primed CTLswere then co-cultured with target T cells from the same donor from whichthe DCs were made. The target T cells were activated using ImmunoCult™ Tcell stimulator (anti-CD2/CD3/CD28, Stem Cell Technologies) in X-VIVO™15 medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/mlIL-2 (Gibco). After 3 days of stimulation, target T cells were eitheredited at the B2M locus using the meganuclease B2M13-14x.479 (B2M KO) oredited at the TRAC locus using TRC1-2L.1592. TRAC-edited target T cellswere transduced with either AAV 7206 (control CAR T) or AAV 7289 (B2MshRNAmiR) at an MOI of 25,000 viral genomes/cell.

B2M- or CAR+ fractions were FACS-sorted to >99% purity using aFACSMelody cell sorter (Becton-Dickinson) and anti-B2M-PE (clone TU-99,BD Biosciences), CD3-PE (clone UCHT1, BioLegend), and anti-FMC63-BV421(clone VM16 produced in house). Sorted target cells were labeled with 1μM Cell Trace Violet (ThermoFisher) and placed into culture withalloantigen-sensitized CTLs at effector:target (E:T) ratios ranging from1:5 through 5:1. 18 hours following culture setup, samples were analyzedfor live dye-positive target cells and percent killing was calculated bycomparing the number of surviving target cells to a “no effector”control.

Natural Killer (NK) responses were also measured. NK cells weremagnetically enriched from PBMC samples using a CD56 positive selectionkit (Stem Cell Technologies). Enriched NK cells were co-cultured for 18h with Cell Trace Violet labeled CAR-T cells (produced with AAV6-7206),B2M knockdown CAR-T cells (produced with AAV6-7289 containing a B2MshRNAmiR), or B2M knockout T cells. After 18 h of co-culture, survivingdye-positive target cells were enumerated and percent killing wascalculated.

CAR T cells with normal levels of B2M surface expression were killed byprimed alloantigen-specific CTLs (FIG. 6A). As the E:T ratio increased,the percent killing increased accordingly, with a maximum of 74% killingat a 2:1 E:T ratio. T cells that were genetically edited at the B2Mlocus, and totally lacked surface expression of B2M protein were killedless efficiently than B2M+ controls at each E:T ratio. The levels ofkilling observed in the CAR T cells encoding a B2M shRNAmiR, andexpressing 5-10% of normal B2M surface levels, were likewise killedinefficiently by primed CTLs, and at a lower percentage than thatobserved by B2M KO.

It was further observed that B2M knockout cells were killed by NK cellsin correlation to E:T ratio, reaching a maximum of 50% killing at 5:1E:T ratio (FIG. 6B). CAR T cells with unmanipulated levels of B2Mexpression were not efficiently killed by NK cells, although somebackground killing was observed at the highest E:T ratio. Notably, B2Mknockdown cells were inefficiently killed by NK cells, although somekilling (28%) is observed at the highest E:T ratio.

Genetic ablation of B2M expression with a meganuclease resulted in cellsthat were highly resistant to killing by primed alloantigen-specificCTLs; however, they were readily killed by NK cells. Producing a stableknockdown of endogenous B2M to 5-10% of normal level using the shRNAmiRapproach resulted in cells that were similarly resistant to CTLs, butsubstantially less sensitive to NK cytolysis. This suggests that thesecells would be more likely to evade NK cell killing in vivo thanallogeneic CAR T cells having a complete knockout of B2M.

In addition to shRNAmiR-mediated knockdown of B2M, a second gene editingapproach was evaluated for NK cell evasion. The second approach involvedthe use of an engineered meganuclease to generate a cleavage site withthe B2M gene, and the introduction of a donor template into the cleavagesite. This donor template encoded an HLA class I histocompatibilityantigen, alpha chain E (HLA-E) polypeptide (SEQ ID NO: 66). Whenexpressed on the cell surface, HLA-E is known to bind to the CD94/NKG2 Ainhibitory receptor on NK cells, and has been shown to shield HLA-E⁺cells from NK cell-mediated lysis. Here, it was examined whethertargeted insertion of an HLA-E coding sequence into the B2M gene couldsimultaneously knockout B2M expression, and thus reduce CAR T cellallogenicity, and express HLA-E to inhibit NK cell killing.

In this study, an apheresis sample was drawn from a healthy, informed,and compensated donor, and the T cells were enriched using the CD3positive selection kit II in accord with the manufacturer's instructions(Stem Cell Technologies). T cells were activated using ImmunoCult T cellstimulator (anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1e6 cells were electroporated with 1 ug of RNA encoding theB2M13-14x.479 meganuclease, which recognizes and cleaves the B2M 13-14recognition sequence (SEQ ID NO: 60) in the B2M locus.

Immediately following electroporation, cells were transduced withAAV7346, which encodes an HLA-E fusion protein comprising threepolypeptides joined by glycine-serine linkers (SEQ ID NO: 66). The firstpolypeptide is a nonamer comprising an HLA-G leader peptide (SEQ ID NO:120) followed by a (GGGGS)3 linker (SEQ ID NO: 121). The secondpolypeptide is a full-length codon-optimized human B2M gene (encodingSEQ ID NO: 119) followed by a (GGGGS)4 linker (SEQ ID NO: 122). Thethird polypeptide is the human HLA-E-01:03 sequence (SEQ ID NO: 118).The transgene is under the control of the JeT promoter and is terminatedby a bi-directional poly-A sequence. The transgene is flanked byhomology arms directing the transgene to insert at the B2M13-14 cutsite.

Following at least 6 days of culture in complete X-VIVO15 mediumcontaining 30 ng/ml IL-2, cells were stained with anti-HLA-ABC-PE(BioLegend, clone W6/32—to detect B2M-edited cells) and anti-HLA-E-BV421(BioLegend clone 3D12). Cells were analyzed for knockout/knock-infrequencies using a Beckman-Coulter CytoFLEX-S or LX. HLA-ABC⁻HLAE⁺cells were purified by FACS using a Beckton-Dickinson FACSMelody.Non-transduced HLA-ABC⁻ (B2M-edited, no AAV) were sorted as a control.Sorted cells were measured for susceptibility to killing by CTLs and NKcells as described previously in this example.

T cells transfected with B2M13-14x.479 RNA developed a population thatlacks display of the canonical MHC I proteins HLA-A, B, and C as well asHLA-E (FIG. 7A). When B2M-edited cells are transduced with AAV7346, apopulation of HLA-ABC− cells that expresses high levels of the HLA-Etransgene are visible (FIG. 7B). These populations were sorted to >99%purity (FIG. 8).

As shown in FIG. 9A, CAR T cells with no B2M edits were killed byalloantigen-primed CTLs, and as E:T ratio increases, increased CAR Tkilling was observed (reaching a maximum of approximately 75%). Incontrast, both B2M knockout cells and B2M knockout cells expressing theHLA-E transgene were not killed efficiently (10% or less), and there wasno increase in killing observed as E:T ratio was increased.

As shown in FIG. 9B, when killing by NK cells was measured,B2M-sufficient CAR T cells were not efficiently targeted by NK cells,but B2M knockout T cells were killed efficiently (with a maximum ofapproximately 50%). HLA-E⁺ cells, despite lacking HLA-ABC expressionwere protected from NK cytolysis to an extent comparable to thatobserved in B2M-sufficient cells. These data indicate that geneticdisruption of the endogenous B2M gene protects T cells against cytolysisby alloantigen-primed CTLs, and an HLA-E transgene prevents themissing-self mechanism of NK cytolysis.

Example 5 In Vivo Efficacy of CAR T Cells Having Knockdown of B2M byshRNAmiR and Stability of B2M Knockdown

These studies were conducted to evaluate the efficacy of CD19 CAR Tcells having a knockdown of B2M by a shRNAmiR, and to determine thestability of the B2M knockdown in vivo in comparison to control CD19 CART cells after the cells were exposed to target cells and activated invivo.

Cryopreserved CD3+ T cells were thawed, rested, and activated aspreviously described. On day 3 post-activation, cells wereelectroporated with mRNA encoding the TRAC-specific nuclease(TRC1-2L.1592) and immediately transduced with the 7206 AAV vectorcarrying the CD19-directed CAR sequence or the 7289 AAV vector carryingthe CD19 CAR with a B2M-targeting shRNAmiR encoded in between the stopcodon for the CAR and the polyadenylation sequence. Residual CD3+(unedited) CAR T cells from both groups were depleted using a CD3magnetic enrichment kit and discarded, and cells from both groups werecryopreserved after expansion post-depletion as previously described.

Female NSG mice were inoculated with a NALM-6 human acute lymphoblasticleukemia cell line expressing firefly luciferase one week before beinginjected intravenously (i.v.) with either vehicle control, CD19-directedCAR T cells, or CD19 CAR-B2M shRNAmiR cells at a dose of 5e6 CAR T cellsper mouse (n=5 animals per group).

Luciferase activity was measured in live animals using IVIS Spectrum(Perkin Elmer, Mass.) imaging system equipped with a CCD camera mountedon a light-tight specimen chamber. On the day of imaging, animals wereinjected with Luciferin substrate and placed in anesthesia inductionchamber. Upon sedation, animals were placed in the dorsal position inthe imaging chamber, equipped with stage heated at physiologicaltemperature, for image acquisition at regular intervals post-luciferinsubstrate. The acquisition time was automatically determined byLivinglmage software. Regions of interest were drawn around each mouse,and flux was quantified and reported as photons per second (p/s). Datawas analyzed and exported using Living Image software 4.5.2. (PerkinElmer, Mass.).

For analysis of B2M expression on human T cells present in the mouseblood, blood samples were taken from individual mice at day 14post-administration of CAR T cells. Red blood cells were lysed, andsamples were then washed and stained for the presence of human CD45 andB2M, and analyzed by flow cytometry, as previously described.

As shown in FIG. 10A, mice engrafted with NALM-6 cells and treated withvehicle (black line with triangles) demonstrated increasing tumor growthover the course of the study with a steady increase in total flux overtime as measured by bioluminescence imaging. In contrast, mice treatedwith either CD19-directed CAR T cells (dark gray line with circles) orCD19-directed CAR T cells with an integrated B2M-targeting shRNAmiR(light gray line with triangles) mediated rapid and durable anti-tumoractivity over the course of the study, as observed by decreased totalflux compared to the vehicle treated animals.

At 14 days post-CAR T administration, blood samples were taken toevaluate the expression of B2M on human CD45+ cells. As shown in FIG.10B, the MFI of B2M expression was more than 90% reduced onCD19-directed CAR T cells with an integrated B2M-targeting shRNAmiRcompared to control CD19-directed CAR T cells, indicating that B2MshRNAmiR bearing cells, which have been activated and have mediatedclearance of target cells, still have reduced levels of B2M expressionrelative to control cells that do not have the B2M shRNAmiR integratedinto the genome.

Two groups of CD19-directed CAR T cells were produced, differing only bythe inclusion of a shRNAmiR vector in one of the constructs to enabletargeted knockdown of B2M expression in the cells that express the CAR.CAR T cells from both groups were able to reduce tumor burden in axenograft model of leukemia. Blood samples taken from the mice confirmedthat the human T cells with the B2M shRNAmiR included had lower levelsof B2M expression on the cell surface than T cells in the blood of micefrom the control CD19 CART cell group, indicating that the knockdown ofB2M is stable even in cells that have been activated and have mediatedkilling of target cells in a relevant mouse model of human leukemia.

Example 6 Stable Knockdown of CS1 in CART Cells

These studies were initiated to determine if CS1 could be stably knockeddown using shRNAmiR sequences. Three candidate guide and passengerstrand sequences for a CS1/SLAMF7 shRNAmiR were built into the miR-Escaffold and positioned after the stop codon of a BCMA-specific CAR(comprising a BCMA-specific scFv, CD8 hinge and transmembrane domains,an N6 co-stimulatory domain, and a CD3 zeta intracellular signalingdomain). These were designated constructs 72101, 72102, and 72103.

In this study, an apheresis sample was drawn from a healthy, informed,and compensated donor, and the T cells were enriched using the CD3positive selection kit II in accord with the manufacturer's instructions(Stem Cell Technologies). T cells were activated using ImmunoCult T cellstimulator (anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1e6 cells were electroporated with 1 ug of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence in the TRAC gene. Nucleofection was carried out inthe presence of 2 ug/1e6 cells of linearized DNA encoding the CAR andone of the candidate CS1/SLAMF7 shRNAmiRs. In this experiment a separatesample was nucleofected with TRC1-2L.1592 as above, and a BCMA CARconstruct that does not encode an RNAi feature. Seven days followingnucleofection, cultures were stained with anti-CD3 (clone UCHT1, BDBiosciences), anti-CS1 (clone 162.1, BioLegend) and biotinylated BCMA(ACRO Biosystems) to detect the CAR (counterstained usingstreptavidin-APC or BV421, BioLegend).

By comparing the CS1 surface expression levels on CAR+ cells to levelsdisplayed on CAR− cells in the same culture, relative knockdownmagnitudes could be measured. Constructs 72101 and 72102 did not visiblyalter CS1 expression, although mean fluorescence intensities suggestedknockdown of 26% and 30% respectively. CAR T cells produced using 72103did display visibly lower levels of CS-1, with few high expressors and acalculated 36% knockdown (FIG. 11).

These experiments demonstrated that expression of endogenous CS1 couldbe stably reduced in CART cells. The 72103 candidate will be examinedfurther with the goal of knocking down CS1 on CS1-specific CAR T cellsin order to prevent fratricidal activity during production.

Example 7 Stable Knockdown of Transforming Growth Factor Beta Receptor 2(TGFBR2) in CAR T Cells

These studies were initiated in order to determine if an additionalendogenous gene, TGFBR2, could be stably knocked down using shRNAmiRsequences. Candidate guide and passenger strand sequences for a TGFBR2shRNAmiR were identified and incorporated into the miR-E scaffold, andpositioned after the stop codon of the FMC63-based anti-CD19 CAR (asdescribed in Example 1). The passenger and guide strands of the TGFBR272110 shRNAmiR are set forth as SEQ ID NOs: 27 and 28, respectively. Thepassenger and guide strands of the TGFBR2 72111 shRNAmiR are set forthas SEQ ID NOs: 29 and 30, respectively. The passenger and guide strandsof the TGFBR2 72112 shRNAmiR are set forth as SEQ ID NOs: 31 and 32,respectively. The passenger and guide strands of the TGFBR2 72113shRNAmiR are set forth as SEQ ID NOs: 33 and 34, respectively. Thepassenger and guide strands of the TGFBR2 72114 shRNAmiR are set forthas SEQ ID NOs: 35 and 36, respectively.

An apheresis sample was drawn from a healthy donor, and the T cells wereenriched using the CD3 positive selection kit II in accordance with themanufacturer's instructions (Stem Cell Technologies). T cells wereactivated using ImmunoCult™ T cell stimulator (anti-CD2/CD3/CD28, StemCell Technologies) in X-VIVO™ 15 medium (Lonza) supplemented with 5%fetal bovine serum and 10 ng/ml IL-2 (Gibco).

After 3 days of stimulation, cells were collected and samples of 1×10⁶cells were electroporated with 1 μg of RNA encoding the TRC 1-2L.1592meganuclease, which recognizes and cleaves the TRC 1-2 recognitionsequence in the TRAC gene. Nucleofection was carried out in the presenceof 2 μg/1×10⁶ cells of linearized DNA encoding the CAR and one of thecandidate TGFBR2 shRNAmiRs. In this experiment, a separate sample wasnucleofected with TRC1-2L.1592 as above, and transduced with AAV 7206,which encodes the FMC63 anti-CD19 CAR, but does not contain an RNAifeature.

Cells were analyzed for TGFBR2 expression at d7, 10, and 14post-nucleofection using the anti-TGFBR2 antibody MM0056-4F14 (Abcam),and an anti-mouse kappa light chain secondary antibody conjugated to PE(BioLegend). CAR+ cells were identified using anti-FMC63-Alx647,produced in house. The mean fluorescence intensity (MFI) of TGFBR2signal on the CAR+ was compared to that on the CAR− cells and a percentknockdown was calculated. The frequency of CAR+ events was consistentacross the different constructs, ranging from 4.9% to 6.7% (FIG. 12).

TGFBR2 surface levels varied from sample to sample in the CAR+population, but were relatively consistent in the CAR− population, whichdid not have the CAR-shRNAmiR construct incorporated into the TRAClocus. Sequences 72111, 72112, and 72113 appeared to support the mostrobust knockdown throughout the experiment (measured by TGFBR2 MFI andsummarized below in Table 2). These three sequences were selected forfurther study.

TABLE 2 Knockdown of TGFBR2 by candidate sequences. % knockdown %knockdown Construct (d7) (d14) 7206 0 0 72110 55 63 72111 64 67 72112 6475 72113 65 63 72114 10 52

This screen of TGFBR2-specific shRNAmiR sequences demonstrated that CART cells could be prepared having a stable knockdown of TGFBR2 at variouslevels. Particularly, constructs 72111, 72112, and 72113 supportedrobust reduction of surface TGFBR2 in the cells into which theCAR-shRNAmiR sequence was successfully incorporated.

Additional studies were performed to compare the shRNAmir-mediatedknockdown approach described herein with nuclease-mediated knockout ofthe TGFBR2 gene.

In this study, an apheresis sample was drawn from a healthy, informed,and compensated donor, and the T cells were enriched using the CD3positive selection kit II in accord with the manufacturer's instructions(Stem Cell Technologies). T cells were activated using ImmunoCult T cellstimulator (anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1e6 cells were electroporated with 1 ug of RNA encoding TRC1-2L.1592,or with one of two nuclease candidates targeting the TGFBR2 gene:TGF1-2x.5 (SEQ ID NO: 64) or TGF1-2L.296 (SEQ ID NO: 65). Electroporatedcells were cultured for 6 days in complete X-VIVO15 medium supplementedwith 30 ng/ml IL-2 prior to an analysis of editing efficiency. This wasachieved by staining cells with an anti-TGFβRII antibody (Abcam cloneMM0056-4F14) followed by anti-mouse IgG κ light chain conjugated to PE(BioLegend clone RMK45). Data were acquired on a Beckman-CoulterCytoFLEX-S or LX.

T cells electroporated with TRC1-2L.1592 were immediately transducedwith AAV72112 (encoding the anti-CD19 FMC63 CAR described in Example 1and a TGFBR2-specific shRNAmiR inserted after the CAR stop codon andbefore a polyA sequence) or AAV7206 (encoding a control FMC63 CAR withno RNAi). Following six days of culture, CD3-CAR+ cells were purified byFACS using methods described above. In addition, TGFβRII-cells fromTGF1-2L.296-edited cultures were sorted in this manner. Sorted cellswere rested in serum free medium overnight before being stimulated with500 ng/ml of recombinant human TGFβ1, the ligand for TGFBR2 (PeproTech).Thirty minutes following TGFβ1 addition, cells were harvested andimmediately prepared for staining using Phos-Flow lyse-fix buffer andPhos-Flow Fix-Perm buffer III (BD Biosciences) according to themanufacturer's instructions. Fixed cells were stained withanti-pSMAD2/3-PE (BD Biosciences) and data were acquired on aBeckman-Coulter CytoFLEX-LX.

Mock nucleofected T cells were stained with the anti-TGFβRII antibodyand the secondary antibody or the secondary antibody alone to serve aspositive and negative controls, respectively, for surface detection ofTGFβRII.

Compared to mock nucleofected cells, introduction of TGF1-2x.5 resultedin TGFBR2 knockout in 38.6% of cells in the culture, while introductionof TGF1-2L.296 resulted in a higher knockout frequency of 69% (FIG. 13).

T cells with disruptions in TGFβRII expression (meganuclease versusRNAi) were sorted and stimulated with TGFβ1 to assess phosphorylation ofSMAD2/3, which are downstream signal transducers of the TGFβR. Comparedto TRAC-edited T cells, which exhibit low pSMAD2/3 signal in the absenceof TGFβ1 and higher pSMAD signal after TGFβ1 exposure, TGFBR2 KO cellslargely fail to respond to ligand exposure (FIG. 14). A small populationof events in the TGFBR2-edited sample exhibits SMAD2/3 phosphorylationfollowing cytokine exposure, although this likely represents a sortimpurity. Compared to sorted TGFβRII-sufficient T cells (7206 CAR T),which phosphorylate SMAD2/3 in response to TGFβ1, and to sorted TGFBR2KO cells, which do not, CAR T cells expressing a TGFBR2-directedshRNAmiR phosphorylate SMAD2/3 to a level that spans the rangedelineated by the positive and negative controls (FIG. 15).

A further experiment was carried out to determine whether meganucleasemediated knock out and shRNAmiR mediated knock down of TGFBR2 couldreduce pSMAD 2/3 signaling in BCMA-specific CAR T cells (expressing theBCMA-specific CAR described in Example 6). Four experimental groups ofBCMA CAR T cells were prepared and tested for pSMAD2/3 levels. The firstand second groups included untreated control and TGFβ1 treated BCMA CART cells, respectively. The third and fourth groups included BCMA CAR Tcells treated with TGFβ1 having either TGFBR2 knocked out with the TGF1-2L.296 meganuclease or TGFBR2 knocked down with the 72112 TGFBR2shRNAmiR, respectively.

Each of the groups of CAR T cells were prepared using the TRC1-2L.1592meganuclease, which recognizes and cleaves the TRC 1-2 recognitionsequence, and a DNA construct encoding a BCMA-specific CAR was insertedinto this recognition sequence as described above. BCMA CAR T cellshaving knock out of TGFBR2 were prepared with the TGF 1-2L.296meganuclease and cells having knock down of TGFBR2 were prepared withthe 72112 TGFBR2 shRNAmiR as described above. The respective BCMA CAR Tcell groups were treated with TGFβ1 (500 ng/ml) and all cell groups wereharvested and analyzed as described above. For the TGFBR2 knock outcells, flow cytometry was gated on the TGFBR2-negative cell population.

As shown in FIG. 16, untreated BCMA CAR T cells had low levels ofpSMAD2/3, whereas treatment with TGFβ1 increased these levels. Knock outof TGFBR2 in BCMA CAR T cells with the TGF 1-2L.296 meganucleasedecreased pSMAD2/3 to levels comparable to, or lower than, the untreatedBCMA CAR T cell control. Knock down of TGFBR2 in BCMA CAR T cells withthe 72112 TGFBR2 shRNAmiR also decreased pSMAD2/3 to levels comparableto the untreated BCMA CAR T cell control.

Thus, these studies demonstrated that TGFBR2 disruptions, either by geneediting or shRNAmiR, result in reduced ability of CART cells tophosphorylate SMAD2/3. No SMAD phosphorylation was detected in CAR Tcells with a disabled TGFBR2 gene, while cells expressing a shRNAmiR areheterogeneous, with cells phosphorylating SMAD2/3 to varying degrees.

Example 8 Comparison of TGFBR2 Knockdown Versus Knockout in CAR T Cells

A further study was conducted in which the activity of CAR T cells wasassessed following various alterations to the TGFβ pathway. CAR T cellswere produced from healthy compensated donor T cells using aBCMA-specific CAR (as described in Example 6) inserted into the TRC 1-2site in the TRAC gene (as described elsewhere). In some variants, asequence encoding a shRNAmiR was also introduced at the TRC 1-2 site inthe same cassette as the CAR (positioned between the CAR stop codon anda polyA sequence). In one variant, the shRNAmiR was a TGFBR2-specificshRNAmiR (construct 72154) while in another variant a shRNAmiR that isirrelevant to TGFβ function (targeting B2M—construct 72155) wasintroduced. A third variant of CAR T cells was produced using construct72155, but they were edited at the TGFBR2 locus using the TGF1-2L296nuclease described above in order to knockout the TGFBR2 gene. CAR Tcells in the TGFBR2 KO group contained 60% KO.

Each group of CAR T cells was challenged with a variety of tumortargets: K562 negative control cells, K562 cells transfected to stablyexpress BCMA, and K562 cells stably expressing BCMA and constitutivelysecreting active TGFβ1 (C223S C225S point mutations). In one experiment,CAR T cells and targets were plated at a 1:1 ratio. At the time pointsindicated in FIG. 17, T cells and any surviving targets were enumerated,and fresh targets were added to the culture so that a 1:1 ratio wasre-established at each time point. The number of T cells in culture withrespect to time are shown in FIG. 17. CAR T cells were not observedexpanding in response to negative control K562 cells.

CART cells with normal levels of TGFBR2 expression expanded >15-fold byday 6 of co-culture when challenged with BCMA+ targets, but only 5-foldin the presence of TGFB-secreting target cells. By comparison, TGFBR2knockdown cells expand similarly in response to BCMA+ targets, but areless inhibited by TGFB-secreting targets (15-fold vs. 10-fold). In aseparate experiment, CAR T cells were plated with targets at a 1:9 E:Tratio, and surviving target cells were enumerated on day 5 and day 7. Byday 7, TGFBR2 knockdown cells had virtually eradicated BCMA+ targetcells, regardless of their TGFb-secretion capacity. Control CAR T cellsonly eradicated control BCMA+ targets and did not eliminateTGFb-secreting BCMA+ targets (FIG. 18).

CAR T cells that were edited with TGF1-2L296 nuclease to knockout TGFBR2displayed a functional advantage over control or TGBFRII knockdown CAR Tcells that was not related to TGFb secretion by the target cells. Asshown in FIG. 19, cultures containing TGFBR2 knockout CAR T cellsexhibited continuous expansion for the 17-day duration of theexperiment. This was associated with an elevated and sustained CD4:CD8ratio in the cultures containing TGFBR2 knockout CAR T cells (FIG. 20).

Together, these data indicate that editing with TGF1-2L296, or theinclusion of a TGFBR2-sepcific shRNAmiR, allow CAR T cells to maintainand carry out effector functions in the presence of suppressive amountsof TGFb 1.

Example 9 Comparison of TGFBR2 Knockdown Versus Knockout in CAR T Cells

The CAR T variants described in Example 8 were also challengedrepeatedly (as above) with the multiple myeloma cell line U266, or withU266 cells engineered to secrete active TGFb 1. Consistent with thefindings from the K562 experiments, the peak expansion of control CAR Tcells was approximately 50% reduced in response to TGFb-secreting U266targets compared to control U266 targets (FIG. 21). CAR T cellsexpressing a TGFBR2-specific shRNAmiR were not inhibited byTGFb1-secreting U266 targets. CAR T cells edited with the TGF1-2L296nuclease to knockout TGFBR2 expanded to higher numbers than other CAR Tvariants and maintained high numbers for the 19-day duration of thisexperiment (FIG. 22). This was also accompanied by an elevated frequencyof CD4+ T cells (FIG. 23). The histograms in FIG. 23 were obtained atday 16 of co-culture and show that relative to control CAR T cultures(72154 at approximately 16% CD4+), TGFBR2 knockdown CAR T cultures hadslightly elevated CD4 frequencies (23%) while dual-edited CAR T culturedcontained 63% CD4+ cells.

Additional observations were made regarding the ability of CAR T cellsto eradicate target cells in culture after their peak of expansion. Dotplots showing surviving target cells expressing BCMA (y-axes) or theTGFb-GFP transgene (x-axes) at the day 16 time point appear in FIG. 24.Despite observing a reduction in T cell numbers on day 16 following there-challenge at day 13, both control and TGFBR2 knockdown CAR T cellswere able to eradicate U266 targets. Dual edited CAR T cells, which didnot exhibit a decrease in T cell number, also eradicated U266 targetsbetween day 13 and day 16. Importantly, only CAR T cells with TGFbresistance (expressing the TGFBR2 shRNAmiR or edited with the TGF1-2L296nuclease) were able to eliminate TGFb-secreting U266 targets. ControlCAR T cells became unable to kill TGFb-secreting U266 targets and theygrew to represent over 90% of the co-culture from day 13 to day 16.

These data support the conclusion in Example 8 that CAR T cells withperturbed expression of TGFBR2, through either shRNAmiR knockdown or bygene knockout, can proliferate and eliminate target cells in thepresence of suppressive levels of TGFb cytokine.

Example 10 Stable Knockdown of CD52 in CAR T Cells

These studies were initiated in order to determine if an additionalendogenous gene, CD52, could be stably knocked down using shRNAmiRsequences. The passenger and guide strands of the CD52 72123 shRNAmiRare set forth as SEQ ID NOs: 37 and 38, respectively. The passenger andguide strands of the CD52 72124 shRNAmiR are set forth as SEQ ID NOs: 39and 40, respectively.

An apheresis sample was drawn from a healthy donor, and the T cells wereenriched using the CD3 positive selection kit II in accordance with themanufacturer's instructions (Stem Cell Technologies). T cells wereactivated using ImmunoCult™ T cell stimulator (anti-CD2/CD3/CD28, StemCell Technologies) in X-VIVO™ 15 medium (Lonza) supplemented with 5%fetal bovine serum and 10 ng/ml IL-2 (Gibco). After 3 days ofstimulation, cells were collected and samples of 1×10⁶ cells wereelectroporated with 1 μg of RNA encoding the TRC 1-2L.1592 meganuclease,which recognizes and cleaves the TRC 1-2 recognition sequence in theTRAC gene. Samples of linearized DNA were also added to cells at thetime of nucleofection to a final concentration of 2 μg/1×10⁶ cells. Twodifferent linearized constructs were used in this experiment, each one avariant of 7206 and each one encoding a different CD52-specific shRNAmiRdownstream of the FMC63 CAR gene's stop codon but upstream of the poly-Atranscriptional terminator.

At 10 days following nucleofection, cultures of cells were stained withanti-CD3-BV711 (Clone UCHT1, BD Biosciences), anti-FMC63-Alx647 (cloneVM16, produced in-house), and anti-CD52-PE (Clone 4C8 BD BioSciences).The CD52 intensity was compared between CD3-CAR+ cells and uneditedCD3+/CAR− cells.

At 10 days post-nucleofection, CD52 intensity on TRAC-edited CAR+ cellswas plotted against CD52 intensity on non-edited (CD3+ CAR−) cells inthe same culture. The CAR+ populations (unshaded histograms) fromcultures nucleofected with constructs 72123 and 72124 expressedapproximately one log lower CD52 signal than corresponding referencepopulations (shaded histograms), representing a stable reduction ofapproximately 90% in the CAR+ populations (FIG. 25).

This study demonstrates that the shRNAmiR approach can be leveraged toefficiently and stably knock down the endogenous CD52 protein in a CAR Tcell population by approximately 90%.

Example 11 Multiplex Knockdown of Proteins by shRNAmiRs in CAR T Cells

Further studies were conducted to determine the feasibility of usingshRNAmiR-mediated knockdown in CAR T cells in a multiplex approach. Inthese studies, both B2M and CD52 were targeted for knockdown in the sameT cells by different shRNAmiRs that were stably expressed from thegenome.

For these experiments, an apheresis sample was drawn from a healthydonor, and the T cells were enriched using the CD3 positive selectionkit II in accordance with the manufacturer's instructions (Stem CellTechnologies). T cells were activated using ImmunoCult™ T cellstimulator (anti-CD2/CD3/CD28, Stem Cell Technologies) in X-VIVO™ 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1×10⁶ cells were electroporated with 1 μg of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence in the TRAC gene.

Samples of linearized DNA were also added to cells at the time ofnucleofection to a final concentration of 1 μg/1×10⁶ cells. Threedifferent linearized constructs were used in this experiment, each one avariant of 7206, expressing a JeT-driven anti-CD19 CAR. One sequencereferred to as clone 7290 contains a B2M-specific shRNAmiR in the 3′untranslated region (UTR) of the CAR. A second construct referred to as72124 contains a CD52-specific shRNAmiR at the same location, and athird construct referred to as 72156 contains the CD52 shRNAmiR followeddirectly by the B2M shRNAmiR in the 3′ UTR of the CAR.

After electroporation, cells were incubated for 7 days in completeX-VIVO15 supplemented with 30 ng/ml IL-2. At this time, T cells wereanalyzed for TRAC editing and CAR insertion, as well as CD52 and B2Mexpression as detailed above.

Populations of CAR⁺CD3⁻ cells were compared against non-edited CD3⁺CAR⁻cells in each sample for CD52 and B2M expression. In samples receiving7290 DNA, CAR T cells exhibited a 91% reduction in B2M surface levels(measured by mean fluorescence intensity) compared to CD3+CAR− cells.Both populations displayed equivalent levels of surface CD52. In samplesreceiving 72124 DNA, the CAR+ population exhibited a 90% reduction inCD52 levels compared to the nonedited population but did not exhibitdecreased B2M levels. CAR⁺ cells containing 72156 DNA demonstratedreduced levels of both CD52 (89%) and B2M (88%) relative to thereference population (FIG. 26). Further, it was demonstrated that abiotinylated anti-CD52 reagent could be used in a negative selectionapproach to deplete CAR− cells which still express high levels of CD52,and enrich the population of CAR+ cells which have reduced CD52expression (FIG. 27).

These findings illustrate that two engineered shRNAmiRs, directedagainst two different transcripts, can be delivered to T cells viasingle-copy targeted insertion, and that both shRNAmiR genes canfunction simultaneously with little to no difference in performancerelative to controls expressing only one shRNAmiR gene.

Example 12 Targeted Insertion of Constructs Encoding a CAR, HLA-E, and ashRNAmiR into a Single Genomic Locus

Studies were conducted to further evaluate the use of shRNAmiRconstructs in approaches for improving allogeneic CAR T cell persistenceand reducing their susceptibility to potential NK cell killing. In thesestudies, four candidate constructs were inserted into T cells at the TRC1-2 recognition site using the TRC 1-2L.1592 meganuclease previouslydescribed. Each construct contained a CD19-specific CAR gene (asdescribed in Example 1, but modified to comprise the signal peptide ofSEQ ID NO: 73) for tumor antigen targeting, a B2M-specific shRNAmiR(same used in the 7289 construct, previously described) optimized toreduce MHC I expression and evade alloreactive T cells, and an HLA-Efusion protein gene for inhibiting NK cytolysis. Constructs 73161-73164,shown in FIG. 28, incorporate these three elements in differentconfigurations that vary in terms of promoter usage, and transcriptionaltermination.

Construct 73161 (FIG. 28A) uses a JeT promoter (SEQ ID NO: 67) to driveexpression of the CAR, HLA-E fusion protein (SEQ ID NO: 66), andshRNAmiR genes as a single transcript that is terminated with abidirectional SV40 polyA signal (labeled BipA; SEQ ID NO: 68). TheshRNAmiR has been encoded in a synthetic intron (SEQ ID NO: 69) insertedat an exon junction in the HLA-E 03-01 allele. The intron will bespliced out and processed by nuclear microRNA biogenesis machinery whilethe remainder of the CAR-HLA-E transcript will be exported to thecytosol and translated into proteins. A P2 A/furin site (SEQ ID NO: 70)enables the separation of the CAR and HLA-E fusion polypeptides.

Constructs 73162-164 (FIGS. 28B-28D) do not use a P2 A/furin cleavagesite but rely on separate promoters to drive CAR and HLA-E expression.In 73162, the shRNAmiR intron has been moved into the CAR gene, both ofwhich are controlled by a JeT promoter and terminated by BipA, whileHLA-E is controlled by a separate JeT promoter and terminated by abovine growth hormone (BGH) polyA signal (SEQ ID NO: 71). In the 73163and 73164 constructs, the shRNAmiR intron is again encoded in HLA-E,with CAR and HLA-E expression controlled by separate promoters andterminators. In 73163, the CAR gene is controlled by the JeT promoterand terminated by BipA, while HLA-E expression is controlled by the EF1αcore promoter (SEQ ID NO: 72) and the BGH terminator. In 73164, bothgenes are controlled by separate JeT promoters and either BipA (CAR) orBGH (HLA-E) terminators. In all four of constructs, the CAR genecontains a signal peptide (SEQ ID NO: 73) that was optimized to increaseCAR density on the surface of edited T cells.

Cryopreserved CD3+ T cells were thawed, rested, and activated aspreviously described. On day 3 post-activation, cells wereelectroporated with mRNA encoding the TRAC-specific nuclease (TRC1-2L.1592) and immediately transduced with an AAV vector at an MOI of25000 viral genomes/cell. Cells received AAV7206, or one of 73161-73164.Additionally, other samples of stimulated T cells were electroporatedwith mRNA encoding both TRC 1-2L.1592 and nuclease B2M 13-14x.479, whichtargets the endogenous B2M locus. These cells were transduced withAAV7206 and an AAV7346 (previously described), which encodes aJeT-driven HLA-E gene and directs insertion with homology to regionsflanking the B2M13-14 site. These cells are referred to as doubleknockout, double knock-in (dKO dKI).

At 6 days following editing and AAV transduction, cells were analyzed byflow cytometry for surface expression of CAR, HLA-ABC, HLA-E, and CD3using reagents, hardware, software, and procedures previously described.

The frequency of TRAC-edited CAR T cells, as well as the intensity ofCAR staining, was assessed and is tabulated in the table of FIG. 29.Compared to the 7206 control, which only expresses a CD19-specific CAR,constructs 73161 and 73163 compared favorably, while constructs 73162and 73164 produced CD3−/CAR+ populations but not as efficiently. Allexperimental constructs supported a higher expression level of CAR onthe surface than 7206 (MFI listed in arbitrary fluorescence units aswell as a percentage of the 7206 signal). This is potentially ascribableto improvements made to the leader peptide sequence.

All experimental constructs supported equally efficient knockdown ofHLA-ABC (89-90%, FIG. 30), resulting from expression of the B2MshRNAmiR, and variable levels of HLA-E expression. Notably, the 73162and 73164 constructs gave rise to populations of CD3−/CAR− populationsthat expressed HLA-E. This observation, coupled with the lower relativefrequencies of CD3−/CAR+ cells in these samples, indicate thatincomplete inserts missing either the left half (encoding the CAR) orthe right half (HLA-E) of the vector were produced and packaged into AAVcapsids. Sequence analysis (not shown) confirms that recombinationevents were driven by identical sequences that may be present in thevector. Fragmentation was observed in vectors 73162 and 73164, whichcontain two JeT promoters, while only intact inserts are detected fromsimilarly-sized vectors, such as 73163, which did not contain anyrepeated identical sequences. Efficient knockdown of HLA-ABC alsosuggests that positioning the shRNAmiR in an intron is permissible anddoes not impair RNAi function.

In summary, these studies demonstrate the efficacy of several constructsthat support three different functions in a CAR T cell (i.e., highexpression of a CAR, high expression of HLA-E, and efficient knockdownof HLA-ABC). Both expression of HLA-E and knockdown of B2M (andtherefore, HLA-ABC), can both potentially act to shield the CAR T cellsfrom NK cell killing. Importantly, each of these multi-componentconstructs can be inserted into a single locus in the genome using asingle nuclease, and avoids the need for multiplex gene editing toinsert a CAR gene into the TRAC locus using a first nuclease, and toseparately insert an HLA-E gene into the B2M locus with a secondnuclease. Moreover, the signal intensity of HLA-E staining was found tobe greater in the multigenic experimental samples (constructs73161-73164) than with our previously described dKO dKI cells, where theHLA-E gene is inserted at the B2M13-14 site (see, MFI reported in tableof FIG. 30, 5th column). Knowing that we can achieve protection from NKcytolysis with the HLA-E expression level observed in dKO/dKI cells, weexpect that the HLA-E expression levels supported by constructs 73161and 73163 will confer protection as well.

Finally, these experiments further show that, in order to achieve ahomogeneous vector, AAV preparation, and cell phenotype, intro-molecularhomology-driven recombination events that result in partial vector lossmust be minimized by avoiding the use of identical, repeated sequencesin the transgene.

Example 13 Evaluation of CAR/HLA-E/B2M shRNAmiR Constructs

In this study, CAR T cells were prepared and assessed for surfaceexpression of CAR, HLA-ABC, and HLA-E as described in Example 12. Here,constructs 7206, 73161, or 73163 were inserted into the TRC 1-2 site inthe TRAC gene. Additional control cells (dKO/dKI) were included as acomparison for HLA-ABC and HLA-E expression levels. These controldKO/dKI cells had the CD19-CAR 7206 construct inserted at the TRC1-2site in the TRAC gene and the HLA-E construct inserted at the B2M 13-14site in the B2M gene.

Tabulated flow cytometry data appear in FIG. 31. The frequencies ofCD3-CAR+ cells in cultures prepared with 73161 and 73163 comparedfavorably to the control (7206), as did the frequencies of TRAC-editedcells expressing transgene (KI of KO). The MFI of the CAR signalappeared to be approximately 2.5 times higher on samples generated with73161 and 73163 than on 7206 or dKO/dKI samples. CAR+ cells generatedwith 73161 and 73163 exhibited greater than 90% reduction in HLA-ABC MFIand the majority of them also express HLA-E. Both the frequency and MFIof HLA-E+ CART cells was higher from the 73161 preparations. Takentogether, these results indicate that vectors 73161 and 73163 supporthigher CAR expression than 7206, a high degree of HLA-ABC knockdown, andhigher HLA-E levels than dKO/dKI approaches, which may confer greateravoidance from NK cell killing in vivo.

Example 14 In Vitro Assessment of Protection Against Alloreactive TCells

This study assessed the ability of CAR T cells to escape natural killer(NK) and cytotoxic lymphocyte (CTL) killing when equipped with a B2MshRNAmiR and an HLA-E transgene. First, CART cells were produced usingvectors 7206, 7289, 73161, or 73163 as described elsewhere. B2M KO Tcells and B2M KO/HLA-E KI cells were produced using a B2M-specificmeganuclease and a B2M-specific repair vector encoding the HLA-E fusionprotein driven by a JeT promoter. All CAR T variants were produced fromcells collected from the same donor (HC6366).

Next, naïve T cells from two unrelated donors (K2916 and K3212) weresensitized against H6366 alloantigens. Briefly, monocytes from HC6366were cultured in the presence of recombinant human GM-CSF (800U/ml—PeproTech) and IL-4 (400 U/ml—PeproTech) for 6 days todifferentiate them into dendritic cell-like APCs. APCs were collectedand co-cultured with naïve T cells from K3212 and K2916 at a T:APC ratioof 5:1. IL-2 (Gibco) was added to the culture after 24 h to a finalconcentration of 10 ng/ml. One week after plating,alloantigen-sensitized T cells were collected, and CTLs were enriched byCD4 depletion (CliniMACS CD4 microbeads, Miltenyi). CAR T variants werelabeled with 1 uM CellTrace Violet (Thermo-Fisher) and then plated withalloantigen-primed CTLs from each donor at the ratios indicated in FIG.32. This co-culture was carried out for 20-24 hours at which time thesamples were labeled with 1 ug/ml of propidium iodide (Sigma) and thenumber of live dye-positive cells in each sample were enumerated using aBeckman-Coulter CytoFLEX-S. Percent killing was determined using azero-effector control.

In FIG. 32, killing of CART cells by K3212 (panel A) or K2916 (panel B)CTLs is shown. Extensive killing was observed against control (7206) CART cells. Low E:T ratios (less than 1:1) resulted in 25-50% killing whileE:T above 1 supported maximal killing for this assay: 50-60%. CAR Tcells expressing a B2M-specific shRNAmiR were less susceptible to CTLkilling with a maximum of 20% in 73163 CAR T cells and 10% in 73161 CART cells.

To assess NK activity against CAR T variants, NK cells were magneticallyenriched from PBMCs from the same donor (HC6366) and an unrelated donor(K799) using a CD56 positive selection kit (StemCell Technologies) andcultured for 48 h in 10 ng/ml IL-15 (Gibco). CAR T target cells werelabeled with Cell Trace Violet (as above) and plated with NK cells atthe ratios specified in FIG. 33. Co-cultures were carried out for 4 to 5days in XVIVO15+5% FBS and 10 ng/ml IL-2. Four to five days afterplating, cultures were labeled with 1 ug/ml propidium iodide andsurviving CAR T targets were enumerated as above. B2M KO cells and 7289CAR T cells (shRNAmiR alone) were nearly eradicated (>90% killing) byboth donor-matched and mismatched NK cells, associating killing with thelack of either normal HLA-ABC levels or an HLA-E transgene. NK killingof CAR T cells with normal HLA-ABC expression (7206) was very low (<10%)or not detected. CAR T variants lacking HLA-ABC but expressing an HLA-Etransgene varied in their susceptibility to NK cytolysis. When culturedwith autologous NK cells, 73163 CAR T cells and B2MKO/HLA-E+ cells weremoderately protected (30-40% killing) while 73161 CAR T cells wererobustly protected (<10% killing). In the experiment conducted withallogeneic NK cells, killing of B2MKO/HLA-E+ cells and 73161 cells wasnot detected.

These observations indicate that constructs 73161 and 73163 conferprotection against alloantigen-specific CTLs without sensitizing thecells to NK killing. Furthermore, this protection can be afforded by asingle recombinant AAV vector which is inserted at the TRAC locus usinga single gene edit.

Example 15 In Vivo Activity of B2M shRNAmiR/HLA-E CAR T Cells

In order to demonstrate that inclusion of a shRNAmiR and an HLA-Etransgene does not impair tumoricidal activity of CAR T cells, weperformed an in vivo experiment in which tumor killing was monitoredover time in immunodeficient mice engrafted with NALM/6 leukemia cells.NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ mice (NOD.scid.γ-chain KO or NSG)mice were purchased from the Jackson Laboratory and injected via thetail vein with 5×10⁵ NALM/6 cells expressing firefly luciferace(Imanis). Six days after tumor implantation, animals were injected withd-luciferin and luminescence was measured using the IVIS imager(Perkin-Elmer). After confirming tumor engraftment, mice were treatedwith either 1×10⁶ or 5×10⁶ CAR T cells (produced as described elsewhere)carrying either the 7206 control transgene (CD19 CAR only), the 7289transgene, the 73161 transgene, or the 73163 transgene. One group ofanimals received vehicle control (saline supplemented with 2% humanserum albumin). Mice were monitored longitudinally for luminescence andbody weight. IACUC-approved humane endpoints were used to determinesurvival times. Luminescence measurements appear in FIG. 34 and survivalis plotted in FIG. 35. Animals receiving vehicle control exhibitedincreasing luminescence after treatment until end point was reached atapproximately day 27 after NALM/6 engraftment. Mice receiving CAR Tintervention exhibited temporarily reduced luminescence with a durationand magnitude that is proportional to the CAR T dose. This isaccompanied by survival benefits, with mice receiving 1×10⁶ CAR T cellssurviving 35-42 days and mice receiving 5×10⁶ cells surviving from 52days to beyond 60 days. No significant contributions to luminescence orsurvival between treatment groups were observed other than due to dosesize. This study indicates that CAR T cells produced using constructs73161 and 73163 have comparable efficacy as 7206 CAR T cells.

Example 16 Screening of DCK shRNAmiRs with Non-Viral DNA Transfection ofCAR/shRNAmiR Constructs

These studies were initiated to evaluate different guide and passengersequences as shRNAmiRs to stably knockdown DCK. The goal was todetermine whether knockdown of DCK in CAR T cells would allow forenrichment of the CD3−/CAR+ population in the presence of purinenucleoside analogs, such as fludarabine, which is commonly used in CAR Tlymphodepletion regimens.

The transgene utilized in this study comprised a JeT promoter drivingthe expression of a CD19 CAR (previously described in Example 1) and ashRNAmiR gene as a single transcript, that is terminated with abidirectional SV40 polyA signal. The transgene was flanked on eitherside by homology arms directing the transgene to insert at the TRC1-2cut site in the TRAC gene. For the shRNAmiRs, five DCK guide andpassenger strand sequences were identified and cloned into a miR-Ebackbone and inserted into the CD19-CAR construct between the stop codonof the CAR and the bidirectional SV40 polyA transcriptional terminator.In separate experiments, the DCK shRNAmiRs evaluated in this studyexhibited reductions of 70% (72136), 40% (72137), 35% (72138), 60%(72140), when compared to endogenous DCK levels of control cellsexpressing a CD19 CAR that did not comprise a DCK-targeting shRNAmiR(7206). These DCK shRNAmiR sequences were tested for their ability toenrich for CD3−/CAR+ population when treated with fludarabine.

Cryopreserved CD3+ T cells were thawed, rested. CD3+ T cells wereactivated using ImmunoCult™ T cell stimulator (anti-CD2/CD3/CD28, StemCell Technologies) in X-VIVO™ 15 medium (Lonza) supplemented with 5%fetal bovine serum and 10 ng/ml IL-2 (Gibco). After 3 days ofstimulation, cells were collected and samples of 1×10⁶ cells wereelectroporated with 1 μg of RNA encoding the TRC 1-2L.1592 meganuclease,which recognizes and cleaves the TRC 1-2 recognition sequence in theTRAC gene. The five CAR-shRNAmiR constructs (constructs 72136-72140)were delivered to T cells as linearized DNA (1 μg/1×10⁶ cells.),simultaneously with the TRC 1-2 nuclease RNA during nucleofection.Electroporated cells were cultured in X-VIVO™ 15 medium supplementedwith 5% fetal bovine serum and 30 ng/ml IL-2.

At day 4 post nucleofection, 2.5e5 viable cells were treated with widerange of doses of fludarabine in a 96 round bottom plate; 50 uM, 5 uM,0.5 uM, 0.05 uM 0.005 uM and an untreated control in complete XVIVO™ 15medium supplemented with 10 ng/mL of IL-15, IL-21 (Gibco). Untreatedcontrol received DMSO at volume equal to highest dose of fludarabine. 2days post treatment, the 96 well plate was spun down, media containingdrug was discarded and the cells were moved from 96 well plate to 48well plate and treated with fresh complete XVIVO™ 15 media, cytokines,and fludarabine (except untreated control which did not receive anydrug). On day 6 post treatment with fludarabine, 200 uL of sample wastaken from each well for staining and remaining cells were spun down andtreated with fresh complete XVIVO™ 15 medium supplemented with 10 ng/mLof IL-15, IL-21 (Gibco) and fludarabine (except untreated control whichdid not receive any drug). Day 6 and 10 post treatment with fludarabine,200 uL of samples of the cultures were stained with anti-CD3-PE(BioLegend Clone UCHT1), anti-FMC63-AlexaFluor647 (clone VM16, producedin-house).

FIG. 36 shows the percent KI of KO plotted at different doses offludarabine tested by flow cytometry and is also listed below thecorresponding shRNAmiR in Table 3. Percent KI of KO is defined as: (%CD3−/CAR+)/(% Total CD3−)×100. Tabulated data were acquired at day 10post-treatment with fludarabine. With CAR expressing DCK shRNAmiRs,treatment with increasing doses of fludarabine resulted in enrichment ofCD3−/CAR+ population and can be observed in Table 3 by an increase inPercent KI of KO. Except 72139, all DCK knockdown constructs showed morethan 70% KI of KO at dose 5 uM of fludarabine. Dose 50 uM Fludarabinewas too toxic to the cells. FIG. 37 shows events/uL of CD3−/CAR+population versus concentration of fludarabine in uM and correlates withpercent KI of KO. Highest percent KI of KO and events/uL were seen atdose 5 uM Fludarabine.

TABLE 3 Percent KI of KO seen by knockdown of DCK by candidatesequences, post treatment with fludarabine. Dose of 7206 72136 7213772138 72139 72140 fludarabine (uM) Untreated 23.06 17.71 18.40 16.5815.72 14.46 (DMSO)  0.005 23.32 20.60 21.93 19.15 17.34 17.61  0.0522.71 21.12 23.43 18.69 17.72 16.85  0.5 27.60 39.03 41.25 41.13 27.5733.94  5 33.27 70.70 72.87 80.70 43.69 70.02 50 4.35 43.14 7.69 8.337.49 4.76Candidate sequences encoded in 72136, 72138 were investigated in furtherexperiments using AAV.

This experiment demonstrated that knocking down DCK using a shRNAmiRallowed for enrichment of CD3−/CAR+ cells in the presence offludarabine. Four out of the five DCK shRNAmiR constructs tested by thisnon-viral approach showed that treatment with dose 5 uM of fludarabinefor 10 days resulted in above 70% KI of KO. Fludarabine is commonly usedalong with cyclophosphamide to lymphodeplete patients prior toadministration of CAR T. This study provides a proof-of-concept thatknocking down DCK with a shRNAmiR makes CAR T cells resistant tofludarabine, allowing for their enrichment. Clinically, the inclusion ofa DCK shRNAmiR could allow for the continued administration offludarabine to a patient after administration of allogeneicfludarabine-resistant CAR Ts, thus suppressing the host immune responseand allowing these drug resistant CAR T cells to have greaterproliferation and persistence in vivo. Thus, a potential clinicalbenefit of knocking down DCK could allow for an extended therapeuticwindow of activity of allogeneic drug resistant CAR T cells, while alsopotentially allowing for synergistic anti-tumor activity of fludarabineand the CAR T therapy.

Example 17 Effects of DCK Knockdown by shRNAmiR on CAR T Cell Phenotypeand Anti-Tumor Activity

These studies were initiated to evaluate different guide and passengersequences as shRNAmiRs (constructs 72136 and 72138) to stably knockdownDCK following AAV transduction. The goal was to determine the effect ofDCK knockdown on CAR T cell phenotype and anti-tumor activity.

Cryopreserved CD3+ T cells were thawed, rested, and activated aspreviously described. On day 3 post-activation, cells wereelectroporated with mRNA encoding the TRAC-specific nuclease TRC1-2L.1592 and immediately transduced with an AAV vector at an MOI of20000 viral genomes/cell. Cells received AAV7206, or 72136 or 72138 orno AAV (only received TRC 1-2L.1592). 3 days post transduction, CAR Tphenotype was studied by staining with anti-CD3-PE (BioLegend, CloneUCHT1), anti-FMC63-AlexFluor647 (clone VM16, produced in-house),anti-CD4-BV711 (BioLegend, clone OKT4), anti-CD8a-BV785 (BioLegend,clone SK1), anti-CD62L-BS515 (BD Pharmingen, clone DREG-56),anti-CD45RO-PE/Cyanine7 (BioLegend, clone UCHL1), anti-CD27-BV421(BioLegend, clone O323). Samples were tested by flow cytometry and datawas acquired on CytoFLEX-S.

2.5e5 viable cells were treated with the following doses of fludarabinein a 48 well plate; 12 uM, 6 uM, 3 uM and an untreated control incomplete Xuri medium supplemented with 10 ng/mL of IL-15, IL-21 (Gibco).Note: Untreated control received DMSO at volume equal to highest dose offludarabine. Day 4 post treatment, cell counts were taken (shown in FIG.38) and 1e6 viable cells were spun down, supernatant was discarded(except samples TRC and 7206 treated with 6 uM and 12 uM fludarabine),and the cells were moved to a new 48 well plate and treated with freshcomplete Xuri media, cytokines, +/− fludarabine. On Day 8 post treatmentwith fludarabine, samples were taken for CAR T phenotype staining asmentioned above. Remaining cells were CD3 depleted using EasySep HumanRelease CD3 positive selection kit. CD3-fractions were retained andcultured in complete Xuri media supplemented with 10 ng/mL of IL-15,IL-21 (Gibco) and no further treatment with fludarabine. Day 14 posttransduction, samples were taken for CAR T phenotype staining prior tosetting up ACEA assay (Refer panel as described above). This is becausethe % CD3−CAR+ is required to determine the exact number of CAR T to addas effectors. Day 15 post transduction, ACEA killing assay was setupusing 72136 or 72138 (DCK knockdown CD19-CAR T) or 7206 (CD19 CAR T)untreated or treated with fludarabine at 3 or 6 uM as effector cells andHEK 293 expressing CD19 as target cells. Triton X-100 was used as apositive control for cytolysis and TRC was used as a negative control.Experiment was setup at Effector:Target (E:T) ratio of 2:1, 1:2 and 1:4.

CAR T phenotype on day 3 post-transduction (pretreatment withfludarabine) was tested via flow cytometry and is summarized in Table 4below. Percent KI of KO is defined as (% CD3−/CAR+)/(% Total CD3−)*100.CD4:CD8 ratios and frequencies for T naïve (Tn), T central memory(Tcm)/T transition memory (Ttm), T effector memory (Tem) are reported inTable 4. Tn are CD62L+CD45RO− (low), whereas Tem are CD62L+, CD45RO+(mid), Ttm are CD62L+, CD45RO+ (high), Tem are CD62L−, CD45RO+. The CART phenotype before treatment with fludarabine remains unchanged between7206 (CD19-CAR) and 72136, 72138 (CD19-CAR expressing DCK shRNAmiRs) asshown below.

TABLE 4 CAR T phenotype based on CD45RO and CD62L gating (pretreatmentwith fludarabine) T cell phenotype derived by staining and gating withCD62L, Percent CD45RO KI of CD4 CD8 AAV KO CD4:CD8 Tn Tcm Ttm Tem Tn TcmTtm Tem  7206 67.87 1.1 0.74 83.56 11.55 2.88 2.01 94.74 3.22 0.55 7213669.31 1.2 0.56 84.22 11.17 2.94 1.98 95.65 2.86 0.55 72138 68.25 1.19 0.7  84.66 10.98 2.53 2.65 95.76 2.92 0.38

FIG. 38 shows viable cell count/mL taken on day 4 post treatment withfludarabine. With TRC only (no AAV) or 7206 (CD19-CAR AAV) a 50% orhigher decrease in cell count was seen post treatment with increasingdose of fludarabine compared to 72136 and 72138 (CD19-CAR AAV expressingDCK shRNAmiRs).

CAR T phenotype on day 8 post treatment with fludarabine was tested viaflow cytometry and is reported in Table 5 below. FIG. 39 shows viablecell counts/mL taken on day 8 post treatment with fludarabine. Due toDCK knockdown, the CAR T cells expressing DCK shRNAmiRs were more viablein the presence of fludarabine compared to TRC nuclease only (no AAV)and 7206 (CD19-CAR AAV). With CAR T expressing DCK shRNAmiRs, treatmentwith increasing doses of fludarabine resulted in enrichment of CD3−/CAR+population and can be observed in Table 5 by an increase in Percent KIof KO. CD4:CD8 ratios and frequencies for T naïve (Tn), T central memory(Tcm)/T transition memory (Ttm), T effector memory (Tem) are alsoreported. No significant differences were observed in CAR T phenotype ofCD19-CAR expressing DCK shRNAmiR's between untreated or samples treatedwith fludarabine. However, with the CD19 CAR (7206) the CD4 and CD8phenotype upon treatment with fludarabine shifts from central memory totransition and effector memory phenotype. This shows that DCK knockdownand treatment with fludarabine did not have any significant effect onthe CAR T cell phenotype.

TABLE 5 CAR T phenotype based on CD45RO and CD62L gating (D8 posttreatment with fludarabine) T cell phenotype derived by staining andgating with Percent CD62L, CD45RO KI of CD4 CD8 AAV KO CD4:CD8 Tn TcmTtm Tem Tn Tcm Ttm Tem 7206 74.09 0.63 0.2  60.15 28.29 8.56 0.86 96.192.31 0.53 untreated 7206 3 uM 72.51 0.68 0.04 25.78 56.91 13.55 0.2886.38 9.76 1.79 Flu 7206 6 uM 74.21 1.7 0.05 16.42 60.53 20.22 0.0974.18 16.42 4.29 Flu 72136 69.27 0.7 0.34 55.95 29.5  11.17 1.61 95.4 2.36 0.89 untreated 72136 3 uM 70.59 0.6 0.8  60.09 25.84 10.6 3.4496.93 1.52 0.49 Flu 72136 6 uM 75.12 0.61 0.48 55.49 29.42 11.5 2.3695.83 2.17 0.64 Flu 72138 72.70 0.66 0.14 56.24 32.47 8.75 0.53 95.523.02 0.6  untreated 72138 3 uM 80.38 0.53 0.23 55.18 28.72 11.35 1.8896.9  1.79 0.48 Flu 72138 6 uM 84.15 0.54 0.19 51.29 32.74 12.01 1.3396.21 2.46 0.56 Flu

FIG. 40 shows % cytolysis (normalized to TRC) using 7206 (CD19-CAR),untreated or treated with 3 and 6 uM fludarabine, as effector cells inan ACEA assay. HEK 293 expressing CD19 were used as target cells.Experiment was setup at E:T ratio of 2:1, 1:2 and 1:4. Triton was usedas a positive control for cytolysis and TRC was used as a negativecontrol. Irrespective of +/− treatment with fludarabine, most efficientkilling was seen at E:T ratio of 2:1. At E:T ratios of 1:2, 1:4, lessefficient killing was seen in presence of increasing doses offludarabine compared to untreated.

FIG. 41 shows % cytolysis (normalized to TRC) using 72138 (CD19-CARexpressing DCK shRNAmiR), untreated or treated with 3 uM or 6 uMfludarabine, as effector cells in an ACEA assay. HEK 293 expressing CD19were used as target cells. Experiment was setup at E:T ratio of 2:1, 1:2and 1:4. Triton was used as a positive control for cytolysis and TRC wasused as a negative control. It was observed that irrespective oftreatment with or without fludarabine, most efficient killing was seenat E:T ratio of 2:1 followed by 1:2 and then 1:4. FIG. 42 shows %cytolysis (normalized to TRC) using 72136 (CD19-CAR expressing DCKshRNAmiR), untreated or treated with 3 uM or 6 uM fludarabine, aseffector cells in an ACEA assay. HEK 293 expressing CD19 were used astarget cells. Experiment was setup at E:T ratio of 2:1, 1:2 and 1:4.Triton was used as a positive control for cytolysis and TRC was used asa negative control. It was observed that irrespective of treatment withor without fludarabine, most efficient killing was seen at E:T ratios of2:1 followed by 1:2 and then 1:4.

Finally, the CD19-CAR expressing DCK shRNAmiRs (72136, 72138) showedmore efficient killing at E:T ratio of 1:2 than CD19 CAR when treatedwith fludarabine. This demonstrated that DCK knockdown using a shRNAmiRand treatment with fludarabine had a synergistic effect on theanti-tumor activity of CD19-CAR.

Example 18 Screening of Glucocorticoid Receptor shRNAmiRs with Non-ViralDNA Transfection of CAR/shRNAmiR Constructs

These studies were initiated to evaluate different guide and passengersequences as shRNAmiRs to stably knockdown the glucocorticoid receptor(GR). The goal was to determine whether knockdown of GR in CAR T cellswould allow for enrichment of the CD3−/CAR+ population in the presenceof corticosteroids such as dexamethasone, which is commonly used in thetreatment of cytokine release syndrome that can be associated with CAR Tcell therapy.

The transgene utilized in this study comprised a JeT promoter drivingthe expression of a CD19-CAR and a shRNAmiR gene as a single transcript,that is terminated with a bidirectional SV40 polyA signal. The transgenewas flanked on either side by homology arms directing the transgene toinsert at the TRC1-2 cut site in the TRAC gene. For the shRNAmiRs, nineGR guide and passenger strand sequences were identified and cloned intoa miR-E backbone and inserted into the CD19-CAR construct between thestop codon of the CAR and the bidirectional SV40 polyA transcriptionalterminator. In separate experiments, the GR shRNAmiRs evaluated in thisstudy exhibited reductions of 37% (72142), 45% (72143), 50% (72145), and56% (72149), when compared to endogenous GR levels of control cells thatexpressed a CD19 CAR but did not comprise a GR-targeting shRNAmiR(7206). These GR shRNAmiR sequences were tested for their ability toenrich for CD3−/CAR+ population when treated with dexamethasone.

Cryopreserved CD3+ T cells were thawed, rested. T cells were activatedusing ImmunoCult™ T cell stimulator (anti-CD2/CD3/CD28, Stem CellTechnologies) in X-VIVO™ 15 medium (Lonza) supplemented with 5% fetalbovine serum and 10 ng/ml IL-2 (Gibco). After 3 days of stimulation,cells were collected and samples of 1×10⁶ cells were electroporated with1 μg of RNA encoding the TRC 1-2L.1592 meganuclease, which recognizesand cleaves the TRC 1-2 recognition sequence in the T cell receptoralpha constant gene. The nine CAR-shRNAmiR constructs (constructs 72142,72143, 72145, 72146, 72148-72152) were delivered to T cells aslinearized DNA (1 μg/1×10⁶ cells.), simultaneously with the TRC1-2nuclease RNA during nucleofection. Electroporated cells were cultured inX-VIVO™ 15 medium supplemented with 5% fetal bovine serum and 30 ng/mlIL-2.

At day 8 post nucleofection, 2.5e5 viable cells were treated with widerange of doses of dexamethasone in a 96 round bottom plate; 100 uM, 10uM, 1 uM, 0.1 uM, 0.01 uM and an untreated control in complete XVIVO™ 15medium supplemented with 10 ng/mL of IL-15, IL-21 (Gibco). Note:Untreated control received 95% ethanol at a volume equal to highest doseof dexamethasone. 3 days post treatment, 96 well plate was spun down,media containing drug was discarded and the cells were moved from 96well plate to 48 well plate and treated with fresh complete XVIVO™ 15media, cytokines and dexamethasone (except untreated control which didnot receive any drug). On day 7 post treatment with dexamethasone, 200uL of sample was taken from each well for staining. Samples were stainedwith anti-CD3-PE (BioLegend, Clone UCHT1), anti-FMC63-AlexaFluor647(clone VM16, produced in-house) and tested by flow cytometry onCytoFLEX-LX.

FIG. 43 shows percent KI of KO at different doses of dexamethasonetested by flow cytometry and is listed below the corresponding shRNAmiRin Table 6. Percent KI of KO is defined as: (% CD3−/CAR+)/(% TotalCD3−)×100. Tabulated data were acquired at day 7 post-treatment withdexamethasone. With CAR expressing GR shRNAmiRs, treatment withincreasing doses of dexamethasone resulted in enrichment of CD3−/CAR+population and can be observed in Table 6 by an increase in percent KIof KO. Except for 72148, all GR knockdown constructs showed between35-50% KI of KO at dose 1, 10 uM of dexamethasone. FIG. 44 showsevents/uL of CD3−/CAR+ plotted versus concentration of dexamethasone inuM. Percent KI of KO correlates with the events/uL.

TABLE 6 Percent KI of KO seen by knockdown of GR by candidate sequences,post treatment with dexamethasone. dexamethasone 7206 72142 72143 7214572146 72148 72149 72150 72151 72152 (uM) Untreated 19.98 25.28 20.415.45 15.23 19.17 12.45 15.99 16.11 16.76 (ethanol)  0.01 15.58 37.2534.3 24.94 26.49 21.98 25.61 23.95 24.75 25.25  0.1 8.55 39.22 41.6933.24 41.85 22.71 40.84 40.33 31.87 33.88  1 16.17 46.84 44.21 40.2738.85 21.69 42.64 39.74 32.54 35.24  10 14.96 44.14 41.4 38.07 39.520.19 43.44 40.58 34.46 34.13 100 13.7 28.77 24.48 23.94 27.25 21.0515.21 23.24 17.49 17.42

This experiment demonstrated that knocking down GR using a shRNAmiRallowed for enrichment of CD3−/CAR+ cells in the presence of acorticosteroid like dexamethasone. Eight out of the nine GR shRNAmiRstested by non-viral approach showed that treatment with dose 1 uM or 10uM of dexamethasone for 7 days resulted in 35-50% KI of KO.

Thus, these experiments confirm that knocking down GR with a shRNAmiRmakes CAR T cells resistant to dexamethasone and allows for theirenrichment. Clinically, corticosteroids are commonly used along withtocilizumab in the treatment of cytokine release syndrome (CRS), apotentially life-threatening toxicity sometimes seen followingadministration of adoptive T-cell therapies for cancer. Cytokine releasesyndrome is associated with elevated circulating levels of severalcytokines. However, the administration of high doses of corticosteroidsmay reduce the clinical effectiveness of CAR T therapy. By making CAR Tcells resistant to corticosteroids, HvG responses against the CAR Tcells can be suppressed without affecting CAR T function, therebyincreasing the potential window for activity and improving safety.

The invention claimed is:
 1. A genetically-modified human T cellcomprising in its genome a cassette comprising the nucleic acid sequenceof SEQ ID NO: 74, wherein said cassette is positioned within a T cellreceptor (TCR) alpha constant region gene.
 2. The genetically-modifiedhuman T cell of claim 1, wherein said cassette is positioned within asequence consisting of SEQ ID NO: 58 within said TCR alpha constantregion gene.
 3. The genetically-modified human T cell of claim 2,wherein said cassette is positioned between nucleotide positions 13 and14 of SEQ ID NO: 58.